Compositions and methods for treatment of celiac disease

ABSTRACT

The invention provides agents and vaccines for treating and diagnosing celiac disease. In particular, the present invention provides a combination of three peptides that are useful for treating and diagnosing celiac disease in a large proportion of patients.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/459,060, which is a continuation of U.S. application Ser. No.13/131,787, now U.S. Pat. No. 8,835,603, issued on Sep. 16, 2014, whichis a national stage filing under 35 U.S.C. §371 of InternationalApplication No. PCT/AU2009/001556, filed Nov. 30, 2009, and which claimsthe benefit of U.S. Provisional Application Ser. No. 61/118,643, filedNov. 30, 2008, the entire contents of each of which are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treatmentof a subject who is sensitive to gluten, in particular a subject who hasceliac disease, and diagnosis thereof and assays and kits for usetherein.

BACKGROUND OF THE INVENTION

Celiac disease, also known as coeliac disease or celiac sprue (coeliacsprue), affects approximately 1% of people in Europe and North America.In many of those affected, celiac disease is unrecognised, but thisclinical oversight is now being rectified with greater clinicalawareness. A gluten free diet is the only current treatment for celiacdisease, and because regular ingestion of as little as 50 mg of gluten(equivalent to 1/100^(th) of a standard slice of bread) damages thesmall intestine, chronic inflammation of the small bowel is commonplacein subjects on a gluten free diet. Persistent inflammation of the smallintestine has been shown to increase the risk of cancer, osteoporosisand death. As gluten is so widely used, for example, in commercialsoups, sauces, ice-creams, etc., maintaining a gluten free diet isdifficult.

Celiac disease occurs in genetically susceptible individuals who possesseither HLA-DQ2 encoded by HLA-DQA1*05 and HLA-DQB1*02 (accounting forabout 90% of individuals), variants of HLA-DQ2, or HLA-DQ8. Suchindividuals mount an inappropriate HLA-DQ2- and/or DQ8-restricted CD4⁺ Tcell-mediated immune response to peptides derived from theaqueous-insoluble proteins of wheat flour, gluten, and related proteinsin rye and barley.

All gluten proteins are considered toxic in celiac disease. In 2006, theNCBI public database Genbank included 345 entries for gluten proteinsfrom bread-making wheat (Triticum aestivum), barley (Hordein vulgare)and rye (Secale cerale).

Predictive approaches have catalogued several hundred distinctputatively “toxic” gluten peptides based upon searches for homologues ofknown epitopes of intestinal T cell clones, or for gluten sequencespredicted or proven to bind to HLA-DQ2 in vitro, having the motiffavouring deamidation by tissue transglutaminase (tTG), and/or sequencesresistant to proteolysis.

Authoritative reviews report there being fifty or so “immunodominant” Tcell epitopes in gluten relevant to celiac disease. However, T cellsraised against hordein or barley have not yet been studied, andHLA-DQ2-restricted T cell epitopes derived from high molecular weight(HMW) glutenin are yet to be defined.

Despite the large number of gluten peptides incriminated in celiacdisease, the protease-resistant α-gliadin 33 merLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO:1; α2-gliadin 56-88)deamidated by tTG: LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:2) iswidely regarded as the optimal stimulatory peptide (for intestinal Tcell lines raised against protease-digested gluten) in HLA-DQ2associated celiac disease. The underlining of Q residues in SEQ ID NO:1,and throughout this disclosure, indicates a glutamine residue amenableto deamidation catalysed by tTG or consistent with the amino-acid motifthat predicts susceptibility to deamidation by tTG, i.e., Q->E.

This α-gliadin 33 mer (SEQ ID NO:1; α2-gliadin 56-88) was recovered froma digestate of the recombinant α2-gliadin, it incorporates multipleoverlapping epitopes previously identified using intestinal T cellclones and lines, and also fresh peripheral blood T cells from HLA-DQ2⁺donors affected by celiac disease after in vivo gluten challenge. Theseepitopes include DQ2-α-I: PFPQPELPY (SEQ ID NO:3); DQ2-α-II: PQPELPYPQ(SEQ ID NO:4); and DQ2-α-III: PYPQPELPY (SEQ ID NO:5). Indeed, in vivogluten challenge in HLA-DQ2⁺ celiac disease patients induces peripheralblood CD4⁺ T cells that are specific for a single 11 mer sequence in theα-gliadin protein sequence, p60-70 PFPQPQLPYPQ (SEQ ID NO:6), that isoptimally bioactive when flanked by three further residues at both theN- and C-terminal, α-gliadin p57-73 QLQPFPQPQLPYPQPQS (SEQ ID NO:7) anddeamidated by tTG or Q65 substituted for glutamate, α-gliadin p57-73QE65 QLQPFPQPELPYPQPQS (SEQ ID NO:8) that includes DQ2-α-I (SEQ ID NO:3)and DQ2-α-II (SEQ ID NO:4). However, there are hundreds of wheat, ryeand barley gluten proteins, and the DQ2-α-I, DQ2-α-II, and DQ2-α-IIIepitopes together typically account for no more than half the toxic Tcell stimulatory properties of gluten in HLA-DQ2⁺ celiac disease.Additional epitopes of relevance to celiac disease are disclosed in WO01/25793, WO 03/104273 and WO 05/105129.

Although T cells have not been raised against barley hordein or ryesecalin, proteins closely related to wheat gluten, the toxicity ofbarley and rye is ascribed to T cells specific for epitopes in wheatgluten, especially DQ2-α-I (SEQ ID NO:3) or DQ2-α-II (SEQ ID NO:4), thatare cross-reactive with related hordein and secalin sequences deamidatedby tTG, in particular PFPQPQQPF (SEQ ID NO:9) deamidated to Hα9/Sα9PFPQPEQPF (SEQ ID NO:10; DQ2-ω-I) or PQPQQPFPQ (SEQ ID NO:11) deamidatedto Hα2/Sα2 PQPEQPFPQ (SEQ ID NO:12), respectively.

Amongst authorities in the field, there is disagreement regarding thedominance, hierarchy, and redundancy of particular peptides in inducingT-cell stimulation in celiac disease.

Understanding the consistency and relative contribution of particularpeptides to the T cell stimulatory capacity of gluten has application.Provided they consistently account for a substantial proportion of the Tcell response to gluten, dominant T cell stimulatory peptides mightalone or collectively enable the development of antigen-specifictherapeutics and diagnostics.

In principle, antigen-specific therapy is an attractive strategy totreat autoimmune and allergic diseases. Whole protein-based approachesto desensitisation are effective for human allergic conditions and alsotreatment and prevention of autoimmunity and allograft rejection inexperimental animal models. However, wider application of protein-basedantigen-specific therapy has been limited by the small but recognisedrisk of anaphylaxis and because relevant antigens may not be suitable aspharmaceuticals or are simply not understood in sufficient detail topermit pharmaceutical development.

The risk of anaphylaxis can be minimised and problems of formulationovercome using short linear, aqueous soluble peptides, encompassingsequences from the disease-relevant antigen recognised by pathogenicCD4⁺ T cells. Peptide-based therapeutic vaccines are effective in inbredmouse models of autoimmunity and allograft rejection in which relevantimmunodominant epitopes and their cognate CD4⁺ T cells are defined.However, even for strongly HLA-associated human immune diseases,identification of pathogenic CD4⁺ T cell epitopes with sufficientconfidence to support rational drug design and pharmaceuticaldevelopment has been very limited.

In many cases, this uncertainty is due to the fact that reported T cellresponses in patients are at the limits of detection, usually dependupon in vitro expansion which may be primary or recall T cell responses,and can often also be found in healthy HLA matched individuals. Thesetechnical challenges have resulted in the compromise that peptideselection for therapeutic vaccines tends to be based upon in vitrobinding affinity for disease-relevant HLA molecules, rather than theirunequivocal definition as epitopes for immunodominant pathogenic Tcells. A further consequence is that peptide-based compounds designed inthis manner tend to encompass an expended cocktail of peptides. It mightbe expected that the larger the cocktail, the greater the likelihood ofdifficulties in formulation, stability and adverse effects, but also themore likely that T cells specific for peptides in the cocktailconsistently make a substantial contribution to the pathogenic T cellresponse in patients.

Given the large number of toxic gluten peptides, the inventors havesought to identify an optimal non-redundant set of immunodominantpeptides from which a minimal mixture could be selected for use in apeptide-based immunotherapy capable of modulating the immune response ofan individual to gluten. The inventors have sought to identifyimmunodominant peptides useful in the treatment of celiac disease byspecifically modifying the pathogenic T cell response to gluten and totherefore provide a vaccine effective against celiac disease. The samepeptide mixture is also useful in diagnosis and monitoringimmunomodulatory therapeutics in celiac disease.

SUMMARY OF THE INVENTION

The present inventors have identified three dominant T cell stimulatorypeptides which together can be used as an agent in an immunotherapy orvaccine to modulate the T cell response to three or more gluten peptidesand to provide tolerance to gluten, allowing treatment of celiacdisease. Accordingly, in one aspect the present invention provides anagent comprising

i) a first peptide comprising the amino acid sequence LQPFPQPELPYPQPQ(SEQ ID NO:13), or a biologically active fragment or variant thereof,

ii) a second peptide comprising the amino acid sequence QPFPQPEQPFPWQP(SEQ ID NO:14), or a biologically active fragment or variant thereof,and

iii) a third peptide comprising the amino acid sequence PEQPIPEQPQPYPQQ(SEQ ID NO:16), or a biologically active fragment or variant thereof.

SEQ ID NO:13 (LQPFPQPELPYPQPQ) encompasses two overlapping epitopes,PFPQPELPY (SEQ ID NO:3) and PQPELPYPQ (SEQ ID NO:4), SEQ ID NO:14(QPFPQPEQPFPWQP) encompasses two overlapping epitopes, PFPQPEQPF (SEQ IDNO:10) and PQPEQPFPW (SEQ ID NO:15; DQ2-ω-II), and SEQ ID NO:16PEQPIPEQPQPYPQQ encompasses the epitope PIPEQPQPY (SEQ ID NO:17;DQ2-Hor-I) and also the predicted epitope EQPIPEQPQ (SEQ ID NO:18)interchangeable with QQPIPEQPQ (SEQ ID NO:19).

In an embodiment, the first, second and/or third peptides comprise an Nterminal acetyl group or pyroglutamate group and/or a C terminal amidegroup. More preferably, the first, second and/or third peptides comprisean N terminal pyroglutamate group and a C terminal amide group.

In a further embodiment, the first, second and/or third peptides areconjugated to a compound. Examples of suitable compounds include, butare not limited to, an adjuvant, and an MHC molecule or binding fragmentthereof.

In a preferred embodiment, each peptide is provided as a separatemolecule. However, in an alternate embodiment, two or three of thefirst, second and third peptides, or biologically active fragment orvariant of one or more thereof, are on a single polypeptide chain.

In a further embodiment, the agent comprises one or more additionalpeptides comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:47, 48, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 95, 102, 103,104, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 136, 169, 170, 171, 172, 173, 174, 177, 178,179, 180, 183, 184, 187, 188, 189, 190, 191, 192, 209, 210, or abiologically active fragment or variant of any one or more thereof.

The additional peptides allow for a wider effective treatment group andgreater breadth of treatment or diagnosis. Particularly, the use ofadditional peptides can increase the likelihood that the agent canabolish inflammation or damage in response to gluten ingestion and allowa celiac disease subject to have a normal diet. Additionally, when theagent is used as a diagnostic, it is advantageous to have more targetsand this is achieved by providing more peptides that might be in theirdeamidated or wild-type form from the list SEQ ID NOs:47, 48, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 75, 76, 77, 78, 79, 80, 81,89, 90, 91, 92, 95, 102, 103, 104, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 136, 169, 170,171, 172, 173, 174, 177, 178, 179, 180, 183, 184, 187, 188, 189, 190,191, 192, 209, 210.

In another aspect, the present invention provides an agent comprisingone or more polynucleotides encoding

i) a first peptide comprising the amino acid sequence LQPFPQPELPYPQPQ(SEQ ID NO:13), or a biologically active fragment or variant thereof,

ii) a second peptide comprising the amino acid sequence QPFPQPEQPFPWQP(SEQ ID NO:14), or a biologically active fragment or variant thereof,

iii) a third peptide comprising the amino acid sequence PEQPIPEQPQPYPQQ(SEQ ID NO:16), or a biologically active fragment or variant thereof,and

iv) optionally one or more additional peptides comprising an amino acidsequence selected from the group consisting of SEQ ID NO:47, 48, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 75, 76, 77, 78, 79, 80, 81,89, 90, 91, 92, 95, 102, 103, 104, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 136, 169, 170,171, 172, 173, 174, 177, 178, 179, 180, 183, 184, 187, 188, 189, 190,191, 192, 209, 210, or a biologically active fragment or variant of anyone or more thereof.

The one or more peptides, or biologically active fragments or variantsthereof, may be encoded by one or more polynucleotides. Thus, at leastsome of the one or more peptides, or biologically active fragments orvariants thereof, may be transcribed and translated from a singlepolynucleotide as a single polypeptide chain.

The agent may also be a mixture of peptides and polynucleotides. Thus,in a further aspect the present invention provides an agent comprising

i) a first peptide as defined herein or a polynucleotide therefor,

ii) a second peptide as defined herein or a polynucleotide therefor, and

iii) a third peptide as defined herein or a polynucleotide therefor. Asthe skilled person would appreciate, one or more of the peptides may bea biologically active fragment or variant of the defined peptidesequence.

In another aspect, the present invention provides a substantiallypurified and/or recombinant peptide comprising, more preferablyconsisting of, an amino acid sequence as shown in any one or more of SEQID NO:16, 69, 73, 75, 78, 80, 87, 91, 92, 95, 96, 98, 100, 104, 107,113, 116, 117, 123, 138, 144, 147, 149, 153, 155, 156, 159, 161, 163,165, 179, 181, 185, 187, 189, 195, 196, 198, 202, 204, 205, 207, 209,215, or 223, or a biologically active fragment or variant of any onefurther or more thereof. In a preferred embodiment of this aspect, thepeptide is 19 amino acids or less in length.

In a further preferred embodiment of the above aspect, the peptidecomprises the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO:16), or abiologically active fragment or variant thereof.

In a further aspect, provided is an isolated and/or exogenouspolynucleotide encoding at least one peptide of the invention.

In a further aspect, provided is a vaccine comprising an agent of theinvention, a peptide of the invention, and/or a polynucleotide of theinvention, and a pharmaceutically acceptable carrier.

In an embodiment, the vaccine comprises an adjuvant.

In another aspect, provided is an isolated antigen presenting cellcomprising an agent of the invention, a peptide of the invention, and/ora polynucleotide of the invention. Examples of antigen presenting celluseful for the invention include, but are not limited to, a dendriticcell, macrophage, B-lymphocyte or a liver sinusoidal endothelial cell.In a preferred embodiment, the antigen presenting cell is a dendriticcell.

In an aspect, provided is a method of modulating a T cell response to agluten peptide in a subject who is sensitive to gluten, the methodcomprising administering to the subject an effective amount of the agentof the invention, the peptide of the invention, the polynucleotide ofthe invention, the vaccine of the invention, and/or the antigenpresenting cell of the invention.

In another aspect, provided is a method of inducing immune tolerance toa gluten peptide in a subject who is sensitive to gluten, the methodcomprising administering to the subject an effective amount of the agentof the invention, the peptide of the invention, the polynucleotide ofthe invention, the vaccine of the invention, and/or the antigenpresenting cell of the invention.

In a further aspect, provided is a method of treating celiac disease,the method comprising administering to a subject who is sensitive togluten an effective amount of the agent of the invention, the peptide ofthe invention, the polynucleotide of the invention, the vaccine of theinvention, and/or the antigen presenting cell of the invention.

In yet a further aspect, provided is a method of modifying cytokinesecretion in a subject who is sensitive to gluten, the method comprisingadministering to the subject an effective amount of the agent of theinvention, the peptide of the invention, the polynucleotide of theinvention, the vaccine of the invention, and/or the antigen presentingcell of the invention.

In one embodiment, interleukin-2 (IL-2), interferon gamma (IFNγ) and/ortumour necrosis factor alpha (TNFα) secretion is reduced. In anotherembodiment, interleukin-10 (IL-10) secretion is increased.

Also provided is the use of the agent of the invention, the peptide ofthe invention, the polynucleotide of the invention, the vaccine of theinvention, and/or the antigen presenting cell of the invention of theinvention for the manufacture of a medicament for modulating a T cellresponse, inducing immune tolerance, treating celiac disease, and/ormodifying cytokine secretion, in a subject who is sensitive to gluten.

In a further aspect, the present invention provides a method fordiagnosing celiac disease in a subject, the method comprising contactinga sample from the subject with the agent of the invention, the peptideof the invention and/or the vaccine of the invention and determining invitro whether one or more of the peptides defined herein bind T cells inthe sample, wherein the binding of one or more of the peptides to Tcells indicates that the subject has, or is susceptible to, celiacdisease.

Also provided is the use of the above diagnostic method to monitorprogression of celiac disease and/or to determine the efficacy of amethod involving administering to the subject who is sensitive to glutenan effective amount of the agent of the invention, the peptide of theinvention, the polynucleotide of the vaccine of the invention and/or theantigen presenting cell of the invention.

In another aspect, the present invention provides a kit for carrying outthe above diagnostic method, the kit comprising the agent of theinvention, the peptide of the invention and/or the vaccine of theinvention, and means to detect binding of one or more of the peptides toT cells. The kit may also include instructions for use. The kit may alsocomprise means for detecting recognition of the agent by T cells.

In a further aspect, the present invention provides a method forproducing the antigen presenting cell of the invention, the methodcomprising

i) obtaining an antigen presenting cell, and

ii) contacting the cell in vitro with the agent of the invention, thepeptide of the invention, the polynucleotide of the invention, and/orthe vaccine of the invention.

Also provided is the use of the agent of the invention, the peptide ofthe invention, the polynucleotide of the invention, the vaccine of theinvention, and/or the antigen presenting cell of the invention indiagnosis or therapy.

In another aspect, the present invention provides a method of making avaccine of the invention, the method comprising combining the first,second and third peptides, and optionally one or more additionalpeptides selected from the group consisting of SEQ ID NOs:47, 48, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 75, 76, 77, 78, 79, 80,81, 89, 90, 91, 92, 95, 102, 103, 104, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 136, 169,170, 171, 172, 173, 174, 177, 178, 179, 180, 183, 184, 187, 188, 189,190, 191, 192, 209, 210, or a biologically active fragment or variant ofany one or more thereof, with a pharmaceutically acceptable carrier andoptionally an adjuvant.

In another aspect, the present invention provides a method ofdetermining whether a composition or food is capable of causing celiacdisease, the method comprising detecting the presence of the agent ofthe invention, the peptide of the invention and/or the polynucleotide ofthe invention in the composition or a food sample.

In a further aspect, the present invention provides a method ofidentifying a protease that can cleave a peptide as defined herein, themethod comprising contacting the peptide with a protease underconditions to effect specific cleavage of the peptide to produce aproteolytic product and detecting the proteolytic product produced.

In another aspect, provided is a method for improving the half lifeand/or bioavailability of a peptide when administered to a subject, themethod comprising modifying the N terminus of the peptide to include anN terminal acetyl or pyroglutamate and modifying the C terminus of thepeptide to include a C terminal amide.

In an embodiment, the peptide is for administering to a subject toinduce immune tolerance.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows the relative frequencies of gluten peptide-specific Tcells detected by IFNγ ELISpot in PBMC collected on day-6 after HLA-DQ2⁺celiac disease donors commence wheat to polymorphisms of α-gliadin 57-73and α-gliadin 57-73 QE65 (SEQ ID NOs:7 and 8, respectively). 1-14 arerepresented by SEQ ID NOs:325, 326, 327, 328, 329, 330, 521, 331, 332,333, 25, 334, 335, and 336, respectively.

FIG. 2: shows IFNγ ELISpot responses of peripheral blood T cells to avariety of T cell epitopes (SEQ ID NOs:350, 351, 352, 353, 354, 44, 355,356, 357, and 358, respectively).

FIG. 3: shows the frequencies of gluten peptide-specific T cellsdetected by IFNγ ELISpot in PBMC collected on Day-6 after HLA-DQ2⁺celiac disease donors commence wheat, rye or barley challenge reveals aclear hierarchy of responses. The 17 merQ is SEQ ID NO:326.

FIG. 4: shows T cells in blood after wheat challenge responded to ahighly consistent hierarchy of gluten peptides. The sequences correspondto SEQ ID NOs:360, 361, 361, 363, 364, 365, 367, 366, 368, and 369 fromtop to bottom, respectively.

FIGS. 5A-5C: show fine-mapping of the immunodominant ω-gliadin peptide,PQQPQQPQQPFPQPQQPFPWQP (SEQ ID NO:52).

FIGS. 6A and 6B: show clear hierarchy of peptides in the Comprehensivepeptide libraries.

FIGS. 7A-C: show the sequences of peptides verified as T-cellstimulatory peptides, their hierarchy, dominance and recognition byT-cell clones raised against the most active peptides after wheat,barley or rye gluten challenge. The 2^(nd) round core sequences in FIG.7A correspond from top to bottom to SEQ ID NOs:370-406. The 2^(nd) roundcore sequences in FIG. 7B correspond from top to bottom to SEQ IDNOs:407-436. The 2^(nd) round core sequences in FIG. 7C correspond fromtop to bottom to SEQ ID NOs:437-465. PFPQPQQPF is SEQ ID NO:9; PFPQPEQPFis SEQ ID NO:10; PQPQQPFPQ is SEQ ID NO:11; PQPEQPFPW is SEQ ID NO:15;PIPEQPQPY is SEQ ID NO:17; QQPQQPFPQ is SEQ ID NO:50; PQPELPYPQ is SEQID NO:246; PFPQPELPY is SEQ ID NO:248; PFPQPQQPI is SEQ ID NO:487;PQPQQPIPV SEQ ID NO:488; PFPQPQLPF is SEQ ID NO:489; PQPQLPFPQ is SEQ IDNO:490; PQQQFPQPQ is SEQ ID NO:491; PFPQPQQPQ is SEQ ID NO:492;QQPQQPFPL is SEQ ID NO:493; QQTFPQQPQ is SEQ ID NO:494; PFPQQPQQPF isSEQ ID NO:495; PFPQPQQTF is SEQ ID NO:496; PFPQQPQQQ is SEQ ID NO:497;QFPQPQQPF is SEQ ID NO:498; PQPFPQQPI is SEQ ID NO:499; PFPQQPIPQ is SEQID NO:500; QQPIPQQPQ is SEQ ID NO:501; QQPFPQQPF is SEQ ID NO:502;PFPQQPFPQ is SEQ ID NO:503; PFPQQPQPY is SEQ ID NO:504; PQPYPQQPQ is SEQID NO:505; PYPQQPQPY is SEQ ID NO:506; QQPFPQQPI is SEQ ID NO:507;PQPQQPIPQ is SEQ ID NO:508; PFPQPQQPT is SEQ ID NO:509; PQPQQPTPI is SEQID NO: 510; IQPQQPFPQ is SEQ ID NO:511; PFPQQPQQI is SEQ ID NO:512;PQPQQQLPL is SEQ ID NO:513; PYPQQPFPQ is SEQ ID NO:514; QQPYPQQPF is SEQID NO:515; PFPQPQLPY is SEQ ID NO:516; PQPQLPYPQ is SEQ ID NO: 517;QQPFPQQPQ is SEQ ID NO:518.

FIG. 8: shows the hierarchy of T-cell stimulatory peptides differsaccording to the whether celiac disease donors undergo wheat, barley orrye challenge. The sequences correspond to SEQ ID NOs:466-486 from topto bottom, respectively.

FIGS. 9A-9D: show that certain mixes of dominant T-cell stimulatorygluten peptides activate substantially larger numbers of T cells inblood collected after in vivo challenge with gluten-containing grains.

FIG. 10: shows the combination of NPL001 (SEQ ID NO:228), NPL002 (SEQ IDNO:229), and NPL003 (SEQ ID NO:230)(NexVax2) activates T cells specificfor NPL001 (SEQ ID NO:228), in the gut (mesenteric lymph nodes, MLN) aswell as spleen and local draining popliteal lymph nodes (PLN) followingsubcutaneous administration to the hind-leg. The proliferation ofNPL001-specific T cells is very similar at the three anatomic sitesdespite the peptides being delivered to the hind-leg. Proliferation of Tcells is dose dependent.

FIGS. 11A and 11B: show repeat administration of NexVax2 (SEQ IDNOs:228, 229 and 230) leads to the reduction in the proportion (FIG.11A) and number (FIG. 11B) of gliadin-specific CD4⁺ T cells in thespleen.

FIGS. 12A and 12B: show repeat administration of NexVax2 leads to theinduction of Treg cells.

FIGS. 13A and 13B: show repeat administration of NexVax2 (SEQ IDNOs:228, 229 and 230) results in an increase in the proportion of IFNγand IL-10 producing cells directly ex-vivo.

FIGS. 14A and 14B: show the proliferative capacity of gliadin-specific Tcells to cognate antigen is diminished following repeat administrationof NexVax2 (SEQ ID NOs:228, 229 and 230) and restored in the presence ofIL-2.

FIGS. 15A and 15B: show T cells from mice treated with NexVax2 (SEQ IDNOs:228, 229 and 230) are able to suppress the proliferation of naïvegliadin-specific T cells.

FIG. 16: shows in vitro cytokine production.

FIGS. 17A and 17B: schedule for dosing, meals and blood collection ofvaccination protocol.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989); T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991); D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996); F. M. Ausubel et al.(editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent); Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988); and J. E. Coligan et al.(editors), Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

As used in the subject specification, the singular forms “a”, “an” and“the” include plural aspects unless the context clearly dictatesotherwise. Thus, for example, reference to “a peptide” includes a singlepeptide, as well as two or more peptides and so forth. Furthermore, anantigen presenting cell is usually provided as a population of suchcells.

The term “celiac disease” refers to a chronic inflammatory disease ofthe small intestine. The disease encompasses a spectrum of conditionscharacterised by varying degrees of gluten sensitivity, including asevere form characterised by a flat small intestinal mucosa(hyperplastic villous atrophy) and other forms characterised by mildersymptoms including fatigue, chronic diarrhoea, malabsorption ofnutrients, weight loss, abdominal distension, anaemia as well as asubstantially enhanced risk for the development of osteoporosis andintestinal malignancies (lymphoma and carcinoma).

The term “sensitive to gluten” refers to the state in which any one ormore of the symptoms of celiac disease or an inappropriate T cellresponse are exhibited by a subject exposed to gluten, or peptidefragment thereof. In a subject who is not sensitive to gluten, there islittle or no T cell response caused by ingestion of gluten. By contrast,in a subject sensitive to gluten there is an inappropriate CD4⁺ T cellmediated immune response to peptides derived from gluten after ingestionthereof.

The terms “immune tolerance”, “immunological tolerance”, “tolerance” or“desensitise” are here defined as to make a sensitised or hypersensitivesubject, less sensitive, insensitive or nonreactive to gluten byreducing the immunological reactivity of a subject towards gluten.Immune tolerance may be generated, for example, by exposure of mucosalsurfaces to tolerance-inducing antigenic fragments of gluten as definedherein. Mucosal administration of both high- and low-dose antigen mayresult in immune tolerance, in which the immune response to subsequentsystemic administration of antigen is reduced. At least two mechanismsof immune tolerance may exist. Tolerance to high-doses of an antigenappears to occur by inactivation or clonal deletion of Th1 and Th2cells. In contrast, tolerance to low doses of antigen leads to bystanderimmune suppression mediated by stimulation of Treg cells to producesuppressive cytokines such as interleukin-4 (IL-4), interleukin-10(IL-10) and TGFβ.

The term “inducing immune tolerance” as used herein refers to bringingabout, producing, or causing immune tolerance to gluten in a subjectsensitive to gluten.

The term “hypersensitive” is here defined as abnormally susceptiblephysiologically to gluten.

The term “anergy” refers to a state of reversible unresponsiveness orhyporesponsiveness of a T cell (or B cell) to an antigen.

As used herein, “Treg” refers to a subclass of T cells whose major roleis to bring T cell-mediated immunity during an immune reaction to anend, and to suppress auto-reactive T cells that escaped negativeselection in the thymus. A “Treg response”, as used herein, ischaracterised by the differentiation and proliferation of the populationof CD4⁺ or CD8⁺ Treg cells which express the forkhead familytranscription factor FOXP3 (forkhead box p3) and/or the MHC Class IIassociated protein LAG-3, and/or express high levels of the IL-2receptor alpha chain (CD25). There is also a minor population of MHCClass I-restricted CD8⁺ FOXP3-expressing Treg cells. The presence ofTreg cells in the peripheral circulation or spleen may be determined byanalysis of CD4⁺/CD25⁺ expression. This may conveniently be achievedusing flow cytometry. In addition, Treg cells may be quantified bydetermining levels of FOXP3 mRNA in peripheral blood- or spleen-derivedmononuclear cells by quantitative reverse transcriptase polymerase chainreaction (PCR). In addition, the induction of a Treg response in vivomay be assessed by the measurement of Treg-associated cytokines fromperipheral blood- or lymph node-derived mononuclear lymphocytes. Tregcells typically show higher expression levels of the anti-inflammatorycytokines such as IL-10 and TGFβ and the presence of these mediators maybe determined by methods known in the art, such as flow cytometry,immunohistochemical staining or ELISA.

The term “T cell stimulatory peptide” or “stimulatory peptide” refers toa peptide or epitope capable of activating a T cell.

The term “activate” or “activating” or “activation” in relation to a Tcell refers to the presentation by an MHC molecule on one cell of anepitope to an appropriate T cell receptor on a second (T) cell, togetherwith binding of a co-stimulatory molecule by the T cell, therebyeliciting a “T cell response”.

As used herein, “toxic peptide” refers to a peptide that stimulates Tcell activation in a subject.

The term “expansion” as used herein refers to the proliferation andamplification of a T cell population following T cell activation.

The term “immunodominant” refers to a subunit of a peptide (epitope)that is most easily recognised by the immune system and thus mostinfluences the specificity of an induced immune response, such as a Tcell response. “Immunodominant” may be used interchangeably with“dominant” herein.

As used herein, the term “modulating a T cell response” refers toregulating or adjusting a T cell response in a subject sensitive togluten, such that the T cell response to gluten is reduced or lessened.

As used herein, “modifying cytokine secretion” refers to changing oraltering somewhat the secretion of cytokines by a subject sensitive togluten, such that the effects of gluten sensitivity in the subject arereduced or lessened. The term encompasses both increased secretion of aparticular cytokine or combination of cytokines and decreased secretionof a particular cytokine or combination of cytokines.

As used herein, “epitope” refers to that portion of an antigen or apeptide that is recognised by the immune system, for example, a T cellreceptor or the major histocompatibility complex (MHC) class I or classII, an antibody, a B cell receptor, which portion is sufficient for highaffinity binding. Generally, a linear epitope for recognition will be atleast about 7 amino acids in length, and may be 8 amino acids, 9 aminoacids, 10 amino acids, or more.

The term “polyepitope” refers to the presence of two or more epitopes(peptides) linked in a single polypeptide chain.

As used herein, “antigen” and “immunogen” and variations thereof aregenerally used interchangeably and refer to the epitope-containingstructure recognised by the immune system.

The term “gluten” or “gluten protein” encompasses alpha (α), beta (β),gamma (γ) and omega (ω) gliadins, and low and high molecular weight (LMWand HMW) glutenins in wheat, B, C and D hordeins in barley, β, γ and ωsecalins in rye, and optionally avenins in oats. “Gluten peptides” arepeptides derived from, or encompassed within, one or more of the glutenproteins.

The term “gliadin” refers to the aqueous alcohol-soluble fraction ofgluten, particularly, but not exclusively, gluten derived from wheat,for example Triticum aestivum.

The term “glutenin” refers to the aqueous alcohol-insoluble fraction ofgluten, particularly but not exclusively, gluten derived from wheat, forexample Triticum aestivum.

As used herein, “hordein” or “barley hordein” refers to gluten derivedfrom barley, Hordein vulgare.

As used herein, “secalin” or “rye secalin” refers to gluten derived fromrye, Secale cerale.

As used herein, “avedin” or “oat avedin” refers to gluten derived fromoats, Avena sativa.

Tissue “transglutaminase” is a crucial factor in celiac disease becauseit promotes gluten-specific T cell responses. Tissue transglutaminasecauses selective deamidation of gluten, which in turn, causes thegeneration of a series of gluten peptides that bind to HLA-DQ2 or -DQ8molecules with high affinity. The resulting HLA-DQ2 (DQ8)-gluten peptideinteraction triggers the proinflammatory CD4 T cell response. Thus, theterm “deamidation” refers to the conversion of glutamine to glutamicacid, or to the conversion of asparagine to aspartic acid. As usedherein, deamidation refers particularly to the conversion of glutamineto glutamic acid in gluten, a process that increases the propensity ofgluten peptides to activate T cells.

The terms “human leukocyte antigen” and “HLA” are here defined as agenetic fingerprint on human white blood cells and platelets, composedof proteins that play a critical role in activating the body's immunesystem to respond to foreign organisms. In humans and other animals, theHLA is also referred to as the “major histocompatibility complex” (MHC).

As used herein, the term “agent” refers to a collection of peptidesand/or polynucleotides. The peptides and/or polynucleotides may be inthe same composition (such as a vaccine), in different compositions or acombination thereof (for example, the first and second peptide definedherein in one composition, and the third in a separate composition). Ifin different compositions, they will preferably be in close proximity,such as in a kit. Accordingly, the methods of the invention contemplateproviding (for example administering to a subject) the individualcomponent peptides and/or polynucleotides of an agent of the inventionin a single composition (vaccine), or sequentially in differentcompositions or a combination thereof.

The term “subject” includes inter alia an individual, patient, target,host or recipient regardless of whether the subject is a human ornon-human animal including mammalian species and also avian species. Theterm “subject”, therefore, includes a human, non-human primate (forexample, gorilla, marmoset, African Green Monkey), livestock animal (forexample, sheep, cow, pig, horse, donkey, goat), laboratory test animal(for example, rat, mouse, rabbit, guinea pig, hamster), companion animal(for example, dog, cat), captive wild animal (for example, fox, deer,game animals) and avian species including poultry birds (for example,chickens, ducks, geese, turkeys). The preferred subject, however, is ahuman, more preferably a human who is HLA-DQ2⁺.

Peptides

The terms “peptide”, “polypeptide”, and “protein” can generally be usedinterchangeably and encompass biologically active fragments, variantsincluding homologues, and salts. However, the term “peptide” istypically used to refer to relatively short molecules comprising lessthan 50, more preferably less than 25, amino acids.

The overall length of each peptide defined herein may be, for example, 7to 50 amino acids, such as 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, or 50 amino acids. It is contemplated thatshorter peptides may prove useful, particularly those that are 20 orfewer amino acids in length, in therapeutics to reduce the likelihood ofanaphylaxis but longer peptides with multiple epitopes are likely to beas effective as multiple short peptides in functional T cell-baseddiagnostics in vitro.

As used herein, a “biologically active fragment” consists of fewer aminoacids than that of the reference peptide defined, for example, by thesequence of SEQ ID NOs:13, 14 or 16. Preferably, biologically activefragments are capable of generating a substantially equal or greater Tcell response in a subject sensitive to gluten as the peptide from whichit is derived. In another embodiment, biologically active fragments arecapable of generating at least 50%, more preferably at least 75% of theT cell response in a subject sensitive to gluten as the peptide fromwhich it is derived. In an embodiment, biologically active fragments are14, 13, 12, 11, 10, 9, 8 and no less than 7 amino acids in length.Deletions and/or additions at either end of any of the peptides areparticularly contemplated.

Examples of biologically active fragments of the peptide provided as SEQID NO:13 are those which include PELP (SEQ ID NO:234), which has beenfound to be essential for T cell recognition.

Accordingly suitable 7 mer fragments of SEQ ID NO:13 include, but arenot limited to:

(SEQ ID NO: 235) QPELPYP; (SEQ ID NO: 236) PELPYPQ; (SEQ ID NO: 237)PQPELPY and (SEQ ID NO: 238) FPQPELP.

Suitable 8 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 239) PELPYPQP; (SEQ ID NO: 240) QPELPYPQ; (SEQ ID NO: 241)PQPELPYP; (SEQ ID NO: 242) FPQPELPY and (SEQ ID NO: 243) PFPQPELP.

Suitable 9 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 244) PELPYPQPQ; (SEQ ID NO: 245) QPELPYPQP; (SEQ ID NO: 246)PQPELPYPQ; (SEQ ID NO: 247) FPQPELPYP; (SEQ ID NO: 248) PFPQPELPY and(SEQ ID NO: 249) QPFPQPELP.

Suitable 10 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 250) QPELPYPQP; (SEQ ID NO: 251) PQPELPYPQP;(SEQ ID NO: 252) PQPELPYPQP; (SEQ ID NO: 253) FPQPELPYPQ;(SEQ ID NO: 254) PFPQPELPYP; (SEQ ID NO: 255) QPFPQPELPY and(SEQ ID NO: 256) LQPFPQPELP.

Suitable 11 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 257) PQPELPYPQPQ; (SEQ ID NO: 258) FPQPELPYPQP;(SEQ ID NO: 259) PFPQPELPYPQ; (SEQ ID NO: 260) QPFPQPELPYP and(SEQ ID NO: 261) LQPFPQPELPY.

Suitable 12 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 262) FPQPELPYPQPQ; (SEQ ID NO: 263) PFPQPELPYPQP;(SEQ ID NO: 264) QPFPQPELPYPQ and (SEQ ID NO: 265) LQPFPQPELPYP.

Suitable 13 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 266) PFPQPELPYPQPQ; (SEQ ID NO: 267) QPFPQPELPYPQP and(SEQ ID NO: 268) LQPFPQPELPYPQ.

Suitable 14 mer fragments of SEQ ID NO:13 include, but are not limitedto:

(SEQ ID NO: 269) QPFPQPELPYPQPQ and (SEQ ID NO: 270) LQPFPQPELPYPQP.

Examples of biologically active fragments of the peptide provided as SEQID NO:14 are those which include QPEQPF (SEQ ID NO:317), which has beenfound to be essential for T cell recognition.

Suitable 7 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 271) QPEQPFP and (SEQ ID NO: 272) PQPEQPF.

Suitable 8 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 273) QPEQPFPW; (SEQ ID NO: 274) PQPEQPFP and(SEQ ID NO: 275) FPQPEQPF.

Suitable 9 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 276) QPEQPFPWQ; (SEQ ID NO: 277) PQPEQPFPW; (SEQ ID NO: 278)FPQPEQPFP and (SEQ ID NO: 279) PFPQPEQPF.

Suitable 10 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 280) QPEQPFPWQP;  (SEQ ID NO: 281) PQPEQPFPWQ; (SEQ ID NO: 282) FPQPEQPFPW;  (SEQ ID NO: 283) PFPQPEQPFP  and (SEQ ID NO: 284) QPFPQPEQPF.

Suitable 11 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 285) PQPEQPFPWQP; (SEQ ID NO: 286) FPQPEQPFPWQ;(SEQ ID NO: 287) PFPQPEQPFPW  and  (SEQ ID NO: 288) QPFPQPEQPFP.

Suitable 12 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 289) FPQPEQPFPWQP;  (SEQ ID NO: 290) PFPQPEQPFPWQ and(SEQ ID NO: 291) QPFPQPEQPFPW.

Suitable 13 mer fragments of SEQ ID NO:14 include, but are not limitedto:

(SEQ ID NO: 292) PFPQPEQPFPWQP  and  (SEQ ID NO: 293) QPFPQPEQPFPWQ.

Examples of biologically active fragments of the peptide provided as SEQID NO:16 are those which include PIPEQPQ (SEQ ID NO:294), which isexpected to be essential for T cell recognition.

Suitable 8 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 295) PIPEQPQP  and  (SEQ ID NO: 296) QPIPEQPQ.

Suitable 9 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 297) PIPEQPQPY; (SEQ ID NO: 298) QPIPEQPQP and (SEQ ID NO: 299) EQPIPEQPQ.

Suitable 10 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 300) PIPEQPQPYP;  (SEQ ID NO: 301) QPIPEQPQPY; (SEQ ID NO: 302) EQPIPEQPQP  and  (SEQ ID NO: 303) PEQPIPEQPQ.

Suitable 11 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 304) PIPEQPQPYPQ;  (SEQ ID NO: 305) QPIPEQPQPYP;(SEQ ID NO: 306) EQPIPEQPQPY and  (SEQ ID NO: 307) PEQPIPEQPQP.

Suitable 12 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 308) PIPEQPQPYPQQ; (SEQ ID NO: 309) QPIPEQPQPYPQ;(SEQ ID NO: 310) EQPIPEQPQPYP  and  (SEQ ID NO: 311) PEQPIPEQPQPY.

Suitable 13 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 312) QPIPEQPQPYPQQ;  (SEQ ID NO: 313) EQPIPEQPQPYPQ and(SEQ ID NO: 314) PEQPIPEQPQPYP.

Suitable 14 mer fragments of SEQ ID NO:16 include, but are not limitedto:

(SEQ ID NO: 315) EQPIPEQPQPYPQQ  and  (SEQ ID NO: 316) PEQPIPEQPQPYPQ.

In one embodiment, the agent or vaccine comprises more than onebiologically active peptide fragment of the peptide of SEQ ID NO:13, 14and/or 16. For example, the peptide of SEQ ID NO:13 could be substitutedfor two separate peptides, one recognised by T cells specific forDQ2-α-I (SEQ ID NO:3) and the other recognised by T cells specific forDQ2-α-II (SEQ ID NO:4).

It has been determined that within the PELP fragment of SEQ ID NO:13essential for T cell recognition, the E must be present or mayoptionally be a D. No other substitution allows for T cell recognition.Accordingly, any variant or fragment of SEQ ID NO:13 must comprise theregion PELP or PDLP.

Biologically active variants include peptides which vary by one or moreamino acids from the defined peptide, which are also known in the art ashomologues. For example, a variant can comprise one or more amino acidsubstitutions in any one or more of the peptides. As used herein,“substituted” or “substitution” includes substitution, replacement,addition, insertion, omission and/or deletion (as such variants may alsobe fragments) of an amino acid residue(s). In particular, this refers topeptides having conservative substitution without losing, orsignificantly diminishing, their use in the methods of the invention.Preferably, biologically active variants are capable of generating asubstantially equal or greater T cell response in a subject sensitive togluten as the peptide from which it is derived. In another embodiment,biologically active variants are capable of generating at least 50%,more preferably at least 75% of the T cell response in a subjectsensitive to gluten as the peptide from which it is derived.

Biologically active variants of the peptides may be identified bymodifying the sequence of each peptide and then assaying the resultingpeptide for the ability to stimulate an immune response, for example,production of T cells.

In an embodiment, no more than 5, more preferably no more than 4, morepreferably no more than 3, more preferably no more than 2, and even morepreferably only 1 amino acid in a defined peptide is varied (bysubstitution, deletion or addition), when compared to a peptide sequencedefined herein.

In an alternate embodiment, the percentage identity between a particularsequence (variant) and a reference sequence (peptide defined herein) isat least about 60% or at least about 70% or at least about 80% or atleast about 90% or at least about 95% or above such as at least about96%, 97%, 98%, 99% or greater. Percentage identity can be determinedusing readily available software packages, such as BLAST(ncbi.nlm.nih.gov/) and GAP.

In one embodiment, the second peptide comprises the amino acid sequencePQQPFPQPEQPFPWQP (SEQ ID NO:320), or a biologically active fragment orvariant thereof.

In another embodiment, the third peptide comprises the amino acidsequence FPEQPIPEQPQPYPQQ (SEQ ID NO:321), or a biologically activefragment or variant thereof.

Natural amino acids include alanine (A), arginine (R), asparagine (N),aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E),glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K),methionine (M), phenylalanine (F), proline (P), serine (S), threonine(T), tryptophan (W), tyrosine (Y), valine (V), hydroxyproline (O and/orHyp), isodityrosine (IDT), and di-isodityrosine (di-IDT).Hydroxyproline, isodityrosine, and di-isodityrosine are formedpost-translationally. Use of natural amino acids, in particular the 20genetically encoded amino acids, is particularly contemplated.

Substitutions may be conservative amino acid substitutions, in which thesubstituted amino acid has similar structural or chemical propertieswith the corresponding amino acid in the reference sequence.Alternatively, the substitutions may be non-conservative amino acidsubstitutions as long as the desired activity is maintained.

By way of example, conservative amino acid substitutions involvesubstitution of one aliphatic or hydrophobic amino acids, for example,alanine, valine, leucine and isoleucine, with another; substitution ofone hydroxyl-containing amino acid, for example, serine and threonine,with another; substitution of one acidic residue, for example, glutamicacid or aspartic acid, with another; replacement of one amide-containingresidue, for example, asparagine and glutamine, with another;replacement of one aromatic residue, for example, phenylalanine andtyrosine, with another; replacement of one basic residue, for example,lysine, arginine and histidine, with another; and replacement of onesmall amino acid, for example, alanine, serine, threonine, methionine,and glycine, with another.

Such conservative substitutions are shown in Table 1 under the headingof preferred substitutions. If such substitutions do not result in achange in functional activity, then more substantial changes, denotedexemplary substitutions in Table 1, may be introduced, and the resultingvariant analysed for functional activity.

TABLE 1 Amino acid substitutions. Original Exemplary Preferred ResidueSubstitutions Substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly(G) Pro Pro His (H) Asn; Gln; Lys;Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu (L)norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala Leu Pro (P) Gly GlySer(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Leu, Ile, Met; Phe; Ala; norleucine Leu

Peptide variants may be produced by mutagenesis or other chemicalmethods. Alanine scanning is a useful technique for identifyingimportant amino acids. In this technique, an amino acid residue isreplaced by Ala and its effect on the peptide's activity is determined.For example, cysteine residues may be substituted to minimisedimerisation via disulfide linkages. Each of the amino acid residues ofthe peptide is analysed in this manner to determine the importantregions of the peptide. Means for preparing such peptides are wellunderstood in the art.

In addition to naturally occurring amino acids, non-naturally occurringamino acids, or modified amino acids, are also contemplated and withinthe scope of the invention. In fact, as used herein, “amino acid” refersto naturally occurring amino acids, non-naturally occurring amino acids,and amino acid analogues, and to the D or L stereoisomers of each.

A non-limiting list of non-conventional and/or non-natural amino acidswhich may be used as suitable substitutions for the naturally occurringamino acids and their standard abbreviations is set out in Table 2.

TABLE 2 Non-conventional amino acids. α-aminobutyric acid Abuα-amino-α-methylbutyrate Mgabu α-methylaminoisobutyrate Maibα-methyl-γ-aminobutyrate Mgabu α-methylcyclohexylalanine Mchexaα-methylcyclopentylalanine Mcpen α-methyl-α-naphthylalanine Manapα-methylpenicillamine Mpen α-naphthylalanine Anap γ-aminobutyric acidGabu aminocyclopropane-carboxylate Cpro aminoisobutyric acid Aibaminonorbornyl-carboxylate Norb cyclohexylalanine Chexacyclopentylalanine Cpen D-alanine Dal D-arginine Darg D-aspartic acidDasp D-cysteine Dcys D-glutamine Dgln D-glutamic acid Dglu D-histidineDhis D-isoleucine Dile D-leucine Dleu D-lysine Dlys D-methionine DmetD-ornithine Dorn D-phenylalanine Dphe D-proline Dpro D-serine DserD-threonine Dthr D-tryptophan Dtrp D-tyrosine Dtyr D-valine DvalD-α-methylalanine Dmala D-α-methylarginine Dmarg D-α-methylasparagineDmasn D-α-methylaspartate Dmasp D-α-methylcysteine DmcysD-α-methylglutamine Dmgln D-α-methylhistidine Dmhis D-α-methylisoleucineDmile D-α-methylleucine Dmleu D-α-methyllysine DmlysD-α-methylmethionine Dmmet D-α-methylornithine DmornD-α-methylphenylalanine Dmphe D-α-methylproline Dmpro D-α-methylserineDmser D-α-methylthreonine Dmthr D-α-methyltryptophan DmtrpD-α-methyltyrosine Dmty D-α-methylvaline Dmval D-N-methylalanine DnmalaD-N-methylarginine Dnmarg D-N-methylasparagine DnmasnD-N-methylaspartate Dnmasp D-N-methylcysteine Dnmcys D-N-methylglutamineDnmgln D-N-methylglutamate Dnmglu D-N-methylhistidine DnmhisD-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysineDnmlys D-N-methylmethionine Dnmmet D-N-methylornithine DnmornD-N-methylphenylalanine Dnmphe D-N-methylproline Dnmpro D-N-methylserineDnmser D-N-methylthreonine Dnmthr D-N-methyltryptophan DnmtrpD-N-methyltyrosine Dnmtyr D-N-methylvaline Dnmval L-t-butylglycine TbugL-ethylglycine Etg L-homophenylalanine Hphe L-methylethylglycine MetgL-norleucine Nle L-norvaline Nva L-α-methylalanine MalaL-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartateMasp L-α-methyl-t-butyglycine Mtbug L-α-methylcysteine McysL-α-methylglutamate Mglu L-α-methylglutamine Mgln L-α-methylhistidineMhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine MileL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylalanine NmalaL-N-methylarginine Nmarg L-N-methylasparagine Nmasn L-N-methylasparticacid Nmasp L-N-methylcysteine Nmcys L-N-methylglutamine NmglnL-N-methylglutamic acid Nmglu L-N-methylhistidine NmhisL-N-methylisoleucine Nmile L-N-methylleucine mleu L-N-methyllysine NmlysL-N-methylmethionine Nmmet L-N-methylnorleucine NmnleL-N-methylnorvaline Nmnva L-N-methylornithine NmornL-N-methylphenylalanine Nmphe L-N-methylproline Nmpro L-N-methylserineNmser L-N-methylthreonine Nmthr L-N-methyltryptophan NmtrpL-N-methyltyrosine Nmtyr L-N-methylvaline Nmval L-N-methylethylglycineNmetg L-N-methyl-t-butylglycine Nmtbug L-N-methylhomophenylalanineNmhphe L-O-methylserine Omser L-O-methylhomoserine OmhserN-(4-aminobutyl)glycine Nglu N-(2-aminoethyl)glycine NaegN-(3-aminopropyl)glycine Norn N-(2,2-diphenylethyl)glycine NbhmN-(3,3-diphenylpropyl)glycine Nbhe N-(3-guanidinopropyl)glycine NargN-(1-hydroxyethyl)glycine Nthr N-(3-indolylyethyl)glycine NhtrpN-(2-carbamylethyl)glycine Ngln N-(2-carboxyethyl)glycine NgluN-(1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine NleuN-(1-methylethyl)glycine Nval N-(2-methylthioethyl)glycine NmetN-amino-α-methylbutyrate Nmaabu N-benzylglycine NpheN-(carbamylmethyl)glycine Nasn N-(carboxymethyl)glycine NaspN-cyclobutylglycine Ncbut N-cycloheptylglycine Nchep N-cyclohexylglycineNchex N-cyclodecylglycine Ncdec N-cylcododecylglycine NcdodN-cyclooctylglycine Ncoct N-cyclopropylglycine NcproN-cycloundecylglycine Ncund N-(hydroxyethyl)glycine NserN-(p-hydroxyphenyl)glycine Nhtyr N-(imidazolylethyl)glycine NhisN-methyl-γ-aminobutyrate Nmgabu N-methylaminoisobutyrate NmaibN-methylcyclohexylalanine Nmchexa N-methylcyclopentylalanine NmcpenN-methylglycine Nala N-methyl-α-naphthylalanine NmanapN-methylpenicillamine Nmpen N-(thiomethyl)glycine Ncys penicillamine PenN-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine NnbheN-(N-(2,2-diphenylethyl)carbamylmethyl)glycine Nnbhm1-carboxy-1-(2,2-diphenylethylamino)cyclopropane Nmbc

Included within the scope of the present invention is an agentcomprising a peptide that is modified during or after translation orsynthesis (for example, by farnesylation, prenylation, myristoylation,glycosylation, palmitoylation, acetylation, phosphorylation (such asphosphotyrosine, phosphoserine or phosphothreonine), amidation,derivatisation by known protecting/blocking groups, proteolyticcleavage, linkage to an antibody molecule or other cellular ligand, andthe like). Any of the numerous chemical modification methods knownwithin the art may be utilised including, but not limited to, specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin, etc.

The phrases “protecting group” and “blocking group” as used herein,refers to modifications to the peptide which protect it from undesirablechemical reactions, particularly in vivo. Examples of such protectinggroups include esters of carboxylic acids and boronic acids, ethers ofalcohols and acetals, and ketals of aldehydes and ketones. Examples ofsuitable groups include acyl protecting groups such as, for example,furoyl, formyl, adipyl, azelayl, suberyl, dansyl, acetyl, theyl,benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromaticurethane protecting groups such as, for example, benzyloxycarbonyl(Cbz); aliphatic urethane protecting groups such as, for example,t-butoxycarbonyl (Boc) or 9-fluorenylmethoxy-carbonyl (FMOC);pyroglutamate and amidation. Many other modifications providingincreased potency, prolonged activity, ease of purification, and/orincreased half-life will be known to the person skilled in the art.

In one embodiment, one of more glutamate residues of one or more of thepeptides may be generated by tTG activity upon a peptide. In alternateembodiment, this reaction occurs in vivo following administration.

The peptides may comprise one or more modifications, which may benatural post-translation modifications or artificial modifications. Themodification may provide a chemical moiety (typically by substitution ofa hydrogen, for example, of a C—H bond), such as an amino, acetyl, acyl,carboxy, hydroxy or halogen (for example, fluorine) group, or acarbohydrate group. Typically, the modification is present on the N- orC-terminal. Furthermore, one or more of the peptides may be PEGylated,where the PEG (polyethyleneoxy group) provides for enhanced lifetime inthe blood stream. One or more of the peptides may also be combined as afusion or chimeric protein with other proteins, or with specific bindingagents that allow targeting to specific moieties on a target cell.

Peptide variants may be obtained in which the peptide has beenchemically modified at the level of amino acid side chains, of aminoacid chirality, and/or of the peptide backbone.

Particular changes can be made to the peptides having SEQ ID NOs:13, 14and/or 16 to improve resistance to degradation or optimise solubilityproperties or otherwise improve bioavailability compared to the parentpeptide, thereby providing peptides having similar or improvedtherapeutic, diagnostic and/or pharmacokinetic properties. A preferredsuch modification includes the use of an N-terminal acetyl group orpyroglutamate and/or a C terminal amide. Such modifications have beenshown in Table 5 which significantly increase the half life andbioavailability of the peptides compared to the parent peptides having afree N and C terminus. Whilst N terminal acetylation and C terminalamidation are suggested in the art in relation to therapeutic peptides,the use of an N-terminal pyroglutamate in the context of inducing immunetolerance has not previously been discussed. It is anticipated thatother peptides useful for inducing immune tolerance could also benefitfrom an N terminal acetyl or pyroglutamate and/or a C terminal amide andaccordingly, in a further aspect there is provided a method forimproving the half life and/or bioavailability of a peptide comprisingmodifying the N terminus of the peptide by the addition of an N terminalacetyl or pyroglutamate and modifying the C terminus of the peptide bythe addition of a C terminal amide. In a particular embodiment, thepeptide comprises the amino acid sequence provided as SEQ ID NOs:228,229 and/or 230.

In one embodiment, the peptide variant of SEQ ID NO:13 has the sequence:

(SEQ ID NO: 228; NPL001) pyroELQPFPQPELPYPQPQ-amide;  or(SEQ ID NO: 231; NPL030) Ac-QLQPFPQPELPYPQPQ-amide.

In another embodiment, the peptide variant of SEQ ID NO:14 has thesequence:

(SEQ ID NO: 229; NPL002) pyroEQPFPQPEQPFPWQP-amide;  or(SEQ ID NO: 232; NPL031) Ac-QQPFPQPEQPFPWQP-amide.

In another embodiment, the peptide variant of SEQ ID NO:16 has thesequence:

(SEQ ID NO: 230; NPL003) pyroEPEQPIPEQPQPYPQQ-amide; or(SEQ ID NO: 233; NPL032) Ac-FPEQPIPEQPQPYPQQ-amide.

The term “pyroE” indicates N-terminal pyroglutamate, and the term “Ac”indicates N-terminal acetyl.

In a particular embodiment, the agent or vaccine comprises NPL001,NPL002 and NPL003. Such agent or vaccine is described herein as NexVax2.

In another embodiment, the peptide variant of SEQ ID NO:13 has thesequence:

(SEQ ID NO: 60; W01-E7) LPYPQPELPYPQ.

In another embodiment, at least one glutamine in any one of the peptidesis substituted by a glutamate.

Certain peptides described herein may exist in particular geometric orstereoisomeric forms. The present invention contemplates all such forms,including cis-(Z) and trans-(E) isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof,and other mixtures thereof, as, falling within the scope of theinvention. Additional asymmetric carbon atoms may be present in asubstituent, such as an alkyl group. All such isomers, as well asmixtures thereof, are intended to be included in this invention.

In another example, to prevent cleavage by peptidases, any one or moreof the peptides may include a non cleavable peptide bond in place of aparticularly sensitive peptide bond to provide a more stable peptide.Such non cleavable peptide bonds may include beta amino acids.

In certain embodiments, any one or more of the peptides may include afunctional group, for example, in place of the scissile peptide bond,which facilitates inhibition of a serine-, cysteine- or aspartate-typeprotease, as appropriate. For example, the invention includes a peptidyldiketone or a peptidyl keto ester, a peptide haloalkylketone, a peptidesulfonyl fluoride, a peptidyl boronate, a peptide epoxide, a peptidyldiazomethane, a peptidyl phosphonate, isocoumarins, benzoxazin-4-ones,carbamates, isocyantes, isatoic anhydrides or the like. Such functionalgroups have been provided in other peptide molecules, and general routesfor their synthesis are known.

A variant may be a mimetic. The term “mimetic” is intended to refer to asubstance which has some chemical similarity to the molecule it mimicsand retains a particular activity of interest (for example, inducingtolerance). The underlying rationale behind the use of peptide mimetics,is that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of T cell and MHC-peptide, antibody and antigen, enzymeand substrate or scaffolding proteins. A peptide mimetic is designed topermit molecular interactions similar to the natural molecule. Mimeticsinclude olefins, phosphonates, aza-amino acid analogues and the like.Persons skilled in the art would readily appreciate methods fordesigning mimetics of peptides and would be able to utilise them todesign mimetics of the peptides defined herein.

The peptides may be analysed by hydrophilicity analysis, which can beused to identify the hydrophobic and hydrophilic regions of the peptide,thus aiding in the design of peptides for experimental manipulation,such as in binding experiments, antibody synthesis, etc. Secondarystructural analysis may also be performed to identify regions of apeptide that adopt specific structural motifs. Manipulation,translation, secondary structure prediction, hydrophilicity andhydrophobicity profiles, open reading frame prediction and plotting, anddetermination of sequence homologies, can be accomplished using computersoftware programs available in the art. Other methods of structuralanalysis including, but not limited to, X-ray crystallography, massspectrometry and gas chromatography, computer modelling, optical rotarydispersion (ORD), or circular dichroism (CD) may also be used.

The peptides, fragments or variants may be in a salt form, preferably, apharmaceutically acceptable salt form. “A pharmaceutically acceptablesalt form” includes the conventional non-toxic salts or quaternaryammonium salts of a peptide, for example, from non-toxic organic orinorganic acids. Conventional non-toxic salts include, for example,those derived from inorganic acids such as hydrochloride, hydrobromic,sulphuric, sulfonic, phosphoric, nitric, and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isothionic, and the like.

The peptides can be provided in the agent or vaccine as separatepeptides or linked, for example, in a polyepitope structure. In oneembodiment, the peptides may be presented in a single polypeptide chain(polyepitope string), i.e., in a linear or circular arrangement. Inanother embodiment, the peptides can be presented in a multiple antigenpresentation system, particularly based on a dendrimer backbone such aspolylysine. A polylysine backbone provides a non-linear, branchedarrangement of epitopes. This system provides the advantage over apolyepitope string that the peptides do not interfere with each other orbe laible to cleavage into cryptic epitopes and thus are able to inducea full T cell response.

Conjugates

One or more of the peptides may be conjugated to a compound usingstandard methods. Examples of compounds to which the peptides can beconjugated include but are not limited to a radioisotope, a fluorescentlabel, a chemiluminescent compound, an enzyme label, a free radical, anavidin-biotin label, a bacteriophage label, a compound that increasesthe half life of the peptide in a subject, an adjuvant, an MHC moleculeor fragment thereof.

The compound may facilitate detection and/or isolation or increaseimmunogenicity of the conjugated peptide.

“Conjugated” as used herein means coupled via covalent or non-covalentbonds. While covalent bonds are preferred, the compound may also belinked to the peptide via complexation without covalent linkage, forexample, via hydrogen bonds or electrostatic, hydrophobic, etc.,interaction.

Typical radioactive isotopes include ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C,⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, and ¹⁵²Eu.

Typical fluorescent labels include fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde,and fluorescamine.

Typical chemiluminescent compounds include luminol, isoluminol, aromaticacridinium esters, imidazoles, acridinium salts, and the oxalate esters.Typical bioluminescent compounds include luciferin, luciferase, andaequorin.

Typical enzyme labels include alkaline phosphatase, beta-galactosidase,glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucoseoxidase, and peroxidase.

In one embodiment, a non-specific linker is included between thecompound and the peptide to which it is conjugated. Such a linker is notinvolved in peptide activity. Rather the linker may serve as a spacerbetween the peptide and a functional moiety. Uses for a linker includeimmobilization of the peptide, such as to aid purification or detection.Alternatively, a linker may allow attachment of a compound to thepeptide that enables specific delivery of the peptide to a particulartarget, such as a cell or tissue, spatially or temporally. When used asa vaccine, one or more of the peptides may be coupled to a linker thatserves as a spacer between the peptide and an immunogenic carrier, orpermits improved coupling between the peptide and the immunogeniccarrier and prevents the formation of cryptic epitopes.

In one embodiment, one or more of the peptides are covalently coupled toan adjuvant (immunogenic carrier protein), such as diphtheria toxoid(DT), keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or thenuclear protein of influenza virus (NP), to increase theirimmunogenicity, using any of several conjugation chemistries known inthe art. A non-specific linker can be present between the peptide andthe immunogenic carrier and is preferably joined to the peptide orco-synthesised to facilitate coupling to the immunogenic carrier and/orto serve as a spacer between the peptide and the immunogenic carrier.

When used as a diagnostic agent, one or more of the peptides arepreferably conjugated to an immunogenic carrier that was not previouslyused for vaccination. When monitoring the success of vaccination, thisprevents the diagnostic agent from reacting to antibodies that wereformed against the carrier fraction of the vaccine.

In one embodiment, the compound is an MHC class II molecule or peptidebinding fragment thereof. The MHC class II molecule may be purified froma biological sample. Alternatively, the MHC class II molecule may berecombinantly produced. A peptide binding fragment of the MHC class IImolecule can be obtained, for example, by enzymatic cleavage of thepurified or recombinant intact molecule. Alternatively, the peptidebinding fragment may be recombinantly produced. In a preferredembodiment, the compound is a recombinant two domain MHC class IImolecule.

In their most basic form, the two domain MHC class II molecule comprisesthe α1 and β1 domain of a mammalian MHC class II molecule wherein theamino terminus of the α1 domain is covalently linked to the carboxyterminus of the β1 domain and wherein the polypeptide does not includethe α2 or β2 domains. The two domain MHC class II molecule is associatedby covalent or non-covalent interaction with a peptide defined herein.In certain embodiments, the peptide is covalently linked to the aminoterminus of the β1 domain of the class II molecule. The two domain MHCclass II molecule may also comprise a detectable label, such as afluorescent label, or a toxin. Where the detectable label or toxin is tobe covalently linked to the MHC molecule in a directed manner (i.e.,rather than being randomly attached) it will generally be linked to thecarboxy terminus of the molecule so as to minimise interference with thepeptide antigen linked at the amino terminus.

In vitro, the two domain MHC class II molecule may be used to detect andquantify T-cells, and regulate T-cell function. Thus, such moleculesloaded with a selected peptide may be used to detect, monitor andquantify the population of T cells that are specific for that peptide.The two domain MHC class II molecule/peptide conjugate may also be usedto induce anergy of gluten-specific T-cells, alleviating symptomsassociated with celiac disease. Alternatively, such molecules may beconjugated with a toxin to more directly kill the disease-causing Tcells. Suitable toxins include protein toxins (for example, ricin,diphtheria, and Pseudomonas toxin), chemotherapeutic agents (forexample, doxorubicin, daunorubicin, methotrexate, cytotoxin, andantisense RNA), antibodies to a cytotoxic T-cell surface molecule,lipases, and radioisotopes emitting “hard”, for example, beta radiation.

Design of Recombinant MHC Class II β1α1 Molecule

The amino acid sequences of mammalian MHC class II α and β chainproteins, as well as nucleic acids encoding these proteins, are wellknown in the art and available from numerous sources including GenBank.

Typically, the α1 domain is regarded as comprising about residues 1-90of the mature α chain. The native peptide linker region between the α1and α2 domains of the MHC class II protein spans from about amino acid76 to about amino acid 93 of the α chain, depending on the particular αchain under consideration. Thus, an al domain may include about aminoacid residues 1-90 of the α chain, but one of skill in the art willrecognise that the C-terminal cut-off of this domain is not necessarilyprecisely defined, and, for example, might occur at any point betweenamino acid residues 70-100 of the α chain. The composition of the α1domain may also vary outside of these parameters depending on themammalian species and the particular α chain in question.

Similarly, the β1 domain is typically regarded as comprising aboutresidues 1-90 of the mature β chain. The linker region between the β1and β2 domains of the MHC class II protein spans from about amino acid85 to about amino acid 100 of the β chain, depending on the particular βchain under consideration. Thus, the β1 protein may include about aminoacid residues 1-100, but one of skill in the art will again recognisethat the C-terminal cut-off of this domain is not necessarily preciselydefined, and, for example, might occur at any point between amino acidresidues 75-105 of the β chain.

When selecting the sequence of a particular domain for inclusion in arecombinant molecule, it is preferable that the entire domain beincluded; to ensure that this is done, the domain sequence may beextended to include part of the linker, or even part of the adjacentdomain. The precise number of amino acids in the α1 and β1 domainsvaries depending on the species of mammal, as well as between classes ofgenes within a species. Rather than a precise structural definitionbased on the number of amino acids, it is the maintenance of domainfunction that is important when selecting the amino acid sequence of aparticular domain. Moreover, one of skill in the art will appreciatethat domain function may also be maintained if somewhat less than theentire amino acid sequence of the selected domain is utilised. Forexample, a number of amino acids at either the amino or carboxy terminiiof the α1 domain may be omitted without affecting domain function.Typically however, the number of amino acids omitted from eitherterminus of the domain sequence will be no greater than 10, and moretypically no greater than 5. Similarly, the α1 and β1 domains mayinclude one or more amino acid sequence variations compared to thenaturally occurring form providing domain function is maintained.

The functional activity of a particular selected domain may be assessedin the context of the peptide loaded MHC class II β1α1 molecule. Forexample, to test a particular β1 domain, it will be linked to afunctional α1 domain, the resultant MHC class II β1α1 molecule peptideloaded and tested for its ability to bind to and/or inhibit antigenspecific T cell function, for example, T cell proliferation.

Nucleic acid molecules encoding these domains may be produced bystandard means, such as amplification by the PCR. Standard approachesfor designing primers for amplifying open reading frames encoding thesedomain may be employed. Libraries suitable for the amplification ofthese domains include, for example, cDNA libraries prepared from themammalian species in question; such libraries are availablecommercially, or may be prepared by standard methods. Thus, for example,constructs encoding the β1 and α1 polypeptides may be produced by PCRusing four primers: primers B1 and B2 corresponding to the 5′ and 3′ends of the β1 coding region, and primers A1 and A2 corresponding to the5′ and 3′ ends of the al coding region. Following PCR amplification ofthe α1 and β1 domain coding regions, these amplified nucleic acidmolecules may each be cloned into standard cloning vectors, or themolecules may be ligated together and then cloned into a suitablevector. To facilitate convenient cloning of the two coding regions,restriction endonuclease recognition sites may be designed into the PCRprimers. For example, primers B2 and A1 may each include a suitable sitesuch that the amplified fragments may be readily ligated togetherfollowing amplification and digestion with the selected restrictionenzyme. In addition, primers B1 and A2 may each include restrictionsites to facilitate cloning into the polylinker site of the selectedvector. Ligation of the two domain coding regions is performed such thatthe coding regions are operably linked, i.e., to maintain the openreading frame. Where the amplified coding regions are separately cloned,the fragments may be subsequently released from the cloning vector andgel purified, preparatory to ligation.

In certain embodiments, a peptide linker is provided between the β1 andα1 domains. Typically, this linker is between 2 and 25 amino acids inlength, and serves to provide flexibility between the domains such thateach domain is free to fold into its native conformation. The linkersequence may conveniently be provided by designing the PCR primers toencode the linker sequence. Thus, in the example described above, thelinker sequence may be encoded by one of the B2 or A1 primers, or acombination of each of these primers.

Variant MHC domain polypeptides may be produced by manipulating thenucleotide sequence of the molecule encoding the domain, for example bysite-directed mutagenesis or the PCR.

Genetic Linkage of Antigenic Peptide to MHC Class II β1α1 Molecule

The MHC Class II β1α1 molecule is used in conjunction with a peptidedefined herein. The MHC Class II β1α1 molecule may be “loaded” with thepeptide in a number of ways, including by covalent attachment of thepeptide to the MHC molecule. This may be conveniently achieved byoperably linking a nucleic acid sequence encoding the selected peptideto the 5′ end of the construct encoding the MHC molecule such that, theexpressed peptide is linked to the N-terminus of β1 of the MHC Class IIβ1α1 molecule. One convenient way of obtaining this result is toincorporate a sequence encoding the peptide into the PCR primers used toamplify the MHC coding regions. Typically, a sequence encoding a linkerpeptide sequence will be included between the molecules encoding theantigenic peptide and the MHC polypeptide. For linking antigens to theMHC polypeptide, the linker should be sufficiently long to permit theantigenic peptide to fit into the peptide groove of the MHC polypeptide.

This genetic system for linkage of the antigenic peptide to the MHCmolecule is particularly useful where a number of MHC molecules withdiffering antigenic peptides are to be produced. The described systempermits the construction of an expression vector in which a uniquerestriction site is included at the 5′ end of the MHC coding region(i.e., at the 5′ end of β1 of the MHC Class II β1α1 molecule). Inconjunction with such a construct, a library of antigenicpeptide-encoding sequences is made, with each antigen-coding regionflanked by sites for the selected restriction enzyme. The inclusion of aparticular antigen into the MHC molecule is then performed simply by (a)releasing the antigen-coding region with the selected restrictionenzyme, (b) cleaving the MHC construct with the same restriction enzyme,and (c) ligating the antigen coding region into the MHC construct. Inthis manner, a large number of MHC-polypeptide constructs can be madeand expressed in a short period of time.

Antigen Loading of Empty β1α1 and α1α2 Molecules

Where the MHC Class II β1α1 molecule is expressed and purified in anempty form (i.e., without attached antigenic peptide), the antigenicpeptide may be loaded into the molecules using standard methods. Suchmethods include simple co-incubation of the purified MHC molecule with apurified preparation of the peptide.

By way of example, empty MHC Class II β1α1 molecules (1 mg/ml; 40 uM)may be loaded by incubation with a 10-fold molar excess of peptide (1mg/ml; 400 uM) at room temperature, for 24 hours. Thereafter, excessunbound peptide may be removed by dialysis against PBS at 4° C. for 24hours. As is known in the art, peptide binding to MHC Class II β1α1molecules can be quantified by silica gel thin layer chromatography(TLC) using radiolabeled peptide. Based on such quantification, theloading may be altered (for example, by changing the molar excess ofpeptide or the time of incubation) to obtain the desired result.

Polynucleotides

The terms “nucleic acid”, “nucleic acid molecule”, “nucleic acidsequence” and “polynucleotide” can generally be used interchangeably andencompass biologically active fragments, and variants includinghomologues.

The overall length of each constituent polynucleotide of an agent maybe, for example, 21 to 150 nucleotides, such as, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 nucleotides.

A “biologically active fragment” of a nucleic acid molecule consists offewer nucleotides than that of the reference peptide encodingpolynucleotide sequence and has a length of at least about 21nucleotides, and may have a length of at least about 35 nucleotides.

The terms “biologically active variant” and“biologically activefragment” have analogous meanings to those ascribed above with regard tothe peptides defined herein.

A “biologically active variant” may comprise a sequence of nucleotideshaving at least 60% identity to the reference peptide encodingpolynucleotide sequence. Percentage identity can be determined usingreadily available software packages, such as BLAST (ncbi.nlm.nih.gov/)and GAP.

Alternatively, or in addition, the “biologically active variant” mayhybridise to the reference peptide encoding nucleotide sequence (or acomplementary form thereof) under low stringency conditions. Referenceherein to “low stringency” refers to at least about 0 to at least about15% v/v formamide and from at least about 1 M to at least about 2 M saltfor hybridization, and at least about 1 M to at least about 2 M salt forwashing conditions. Generally, low stringency is at from about 25-30° C.to about 42° C. The temperature may be altered and higher temperaturesused to replace formamide and/or to give alternative stringencyconditions. Alternative stringency conditions may be applied wherenecessary, such as medium or high stringency. Reference herein to“medium stringency” refers to from at least about 16% v/v to at leastabout 30% v/v formamide and from at least about 0.5 M to at least about0.9 M salt for hybridization, and at least about 0.5 M to at least about0.9 M salt for washing conditions. Reference herein to “high stringency”refers to from at least about 31% v/v to at least about 50% v/vformamide and from at least about 0.01 M to at least about 0.15 M saltfor hybridization, and at least about 0.01 M to at least about 0.15 Msalt for washing conditions.

In general, washing is carried out at Tm=69.3+0.41 (G+C) %. However, theTm of a duplex nucleic acid molecule decreases by 1° C. with everyincrease of 1% in the number of mismatch base pairs. Formamide isoptional in these hybridization conditions.

Particularly preferred levels of stringency are defined as follows: lowstringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; moderatestringency is 2×SSC buffer, 0.1% w/v SDS at 20-65° C.; high stringencyis 0.1×SSC buffer, 0.1% w/v SDS at, at least 65° C.

Biological variants include polynucleotides that vary by one or morenucleotides from the reference polynucleotide. For example, a variantcan comprise a substitution of one or more naturally occurringnucleotides with an analogue (such as the morpholine ring), methylatednucleotide, internucleotide modifications such as uncharged linkages(for example, methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.), charged linkages (for example, phosphorothioates,phosphorodithioates, etc.), pendent moieties (for example,polypeptides), intercalators (for example, acridine, psoralen, etc.),chelators, alkylators and modified linkages (for example, α-anomericnucleic acids, etc.).

Polynucleotides encoding one or more of the peptides may be provided ina vector.

A polynucleotide encoding one or more of the peptides defined herein canbe used for the recombinant production of the peptides using techniqueswell known in the art. Alternatively, the polynucleotide can be used toimmunise/tolerise a subject to gluten.

A polynucleotide for use in the invention includes a DNA sequence thatcan be derived from one or more of the peptides, bearing in mind thedegeneracy of codon usage. This is well known in the art, as isknowledge of codon usage in different expression hosts, which is helpfulin optimizing the recombinant expression of the peptides.

When the polynucleotide is used for the recombinant production of one ormore of the peptides, the polynucleotide may include the coding sequencefor the peptides alone or the coding sequence for the peptides inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro-proteinsequence, linker peptide sequence, or other fusion peptide portions. Forexample, a marker sequence which facilitates purification of the fusedpeptide can be encoded. In certain embodiments, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.), oris an HA tag, or is glutathione-S-transferase. The polynucleotide mayalso contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilise mRNA.

Antigen Presenting Cells

The agent and/or peptides defined herein may be delivered by loadingAPCs with, for example, the first, second and third peptides, abiologically active fragment or variant of one or more thereof, and/or apolynucleotide encoding one or more thereof.

Preferably, the APCs are selected from the group consisting of dendriticcells, macrophages, B-lymphocytes and liver sinusoidal endothelial cellsthat express MHC class II molecules shared with the MHC phenotype of thesubject. For example, the APCs may express HLA-DQ2 (for example, HLADQA1*05 and HLA DQB1*02) and/or HLA DQ8. The APCs employed for thispurpose may be isolated from the subject to whom they are to bedelivered after loading, or they may be obtained from an allo-matchedsubject.

By “loading” an APC it is meant that the APC is incubated or transfectedwith the peptides, a biologically active fragment or variant of one ormore thereof, or a polynucleotide encoding one or more thereof. Loadingan APC can be achieved by using conventional nucleic acid transfectionmethods, such as lipid-mediated transfection, electroporation, andcalcium phosphate transfection.

Peptide Production

The peptides can be prepared in any suitable manner. For example, thepeptides can be recombinantly and/or synthetically produced.

The peptides may be synthesised by standard chemistry techniques,including synthesis by automated procedure using a commerciallyavailable peptide synthesiser. In general, peptide analogues areprepared by solid-phase peptide synthesis methodology which may involvecoupling each protected amino acid residue to a resin support,preferably a 4-methylbenzhydrylamine resin, by activation withdicyclohexylcarbodiimide to yield a peptide with a C-terminal amide.Alternatively, a chloromethyl resin (Merrifield resin) may be used toyield a peptide with a free carboxylic acid at the C-terminal. After thelast residue has been attached, the protected peptide-resin is treatedwith hydrogen fluoride to cleave the peptide from the resin, as well asdeprotect the side chain functional groups. Crude product can be furtherpurified by gel filtration, high pressure liquid chromatography (HPLC),partition chromatography, or ion-exchange chromatography.

If desired, and as outlined above, various groups may be introduced intothe peptide of the agent during synthesis or during expression, whichallow for linking to other molecules or to a surface. For example,cysteines can be used to make thioethers, histidines for linking to ametal ion complex, carboxyl groups for forming amides or esters, aminogroups for forming amides, and the like.

The peptides may also be produced using cell-free translation systems.Standard translation systems, such as reticulocyte lysates and wheatgerm extracts, use RNA as a template; whereas “coupled” and “linked”systems start with DNA templates, which are transcribed into RNA thentranslated.

Alternatively, the peptides may be produced by transfecting host cellswith expression vectors that comprise a polynucleotide(s) that encodesone or more peptides.

For recombinant production, a recombinant construct comprising asequence which encodes one or more of the peptides is introduced intohost cells by conventional methods such as calcium phosphatetransfection, DEAE-dextran mediated transfection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape lading, ballistic introduction or infection.

One or more of the peptides may be expressed in suitable host cells,such as, for example, mammalian cells (for example, COS, CHO, BHK, 293HEK, VERO, HeLa, HepG2, MDCK, W138, or NIH 3T3 cells), yeast (forexample, Saccharomyces or Pichia), bacteria (for example, E. coli, P.pastoris, or B. subtilis), insect cells (for example, baculovirus in Sf9cells) or other cells under the control of appropriate promoters usingconventional techniques. Following transformation of the suitable hoststrain and growth of the host strain to an appropriate cell density, thecells are harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for further purificationof the peptide or variant thereof.

Suitable expression vectors include, for example, chromosomal,non-chromosomal and synthetic polynucleotides, for example, derivativesof SV40, bacterial plasmids, phage DNAs, yeast plasmids, vectors derivedfrom combinations of plasmids and phage DNAs, viral DNA such as vacciniaviruses, adenovirus, adeno-associated virus, lentivirus, canary poxvirus, fowl pox virus, pseudorabies, baculovirus, herpes virus andretrovirus. The polynucleotide may be introduced into the expressionvector by conventional procedures known in the art.

The polynucleotide which encodes one or more peptides may be operativelylinked to an expression control sequence, i.e., a promoter, whichdirects mRNA synthesis. Representative examples of such promotersinclude the LTR or SV40 promoter, the E. coli lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or in viruses. The expressionvector may also contain a ribosome binding site for translationinitiation and a transcription terminator.

The expression vectors may also include an origin of replication and aselectable marker, such as the ampicillin resistance gene of E. coli topermit selection of transformed cells, i.e., cells that are expressingthe heterologous polynucleotide. The nucleic acid molecule encoding oneor more of the peptides may be incorporated into the vector in framewith translation initiation and termination sequences.

One or more of the peptides can be recovered and purified fromrecombinant cell cultures (i.e., from the cells or culture medium) bywell known methods including ammonium sulphate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxyapatite chromatography, lectinchromatography, and HPLC. Well known techniques for refolding proteinsmay be employed to regenerate active conformation when the peptide isdenatured during isolation and or purification.

To produce a glycosylated peptide, it is preferred that recombinanttechniques be used. To produce a glycosylated peptide, it is preferredthat mammalian cells such as, COS-7 and Hep-G2 cells be employed in therecombinant techniques.

The peptides can also be prepared by cleavage of longer peptides,especially from food extracts.

Pharmaceutically acceptable salts of the peptides can be synthesisedfrom the peptides which contain a basic or acid moiety by conventionalchemical methods. Generally, the salts are prepared by reacting the freebase or acid with stoichiometric amounts or with an excess of thedesired salt-forming inorganic or organic acid or base in a suitablesolvent.

Vaccines and Administration

The invention also provides a vaccine comprising the first, second andthird peptides, a biologically active fragment or variant of one or morethereof, and/or a polynucleotide encoding one or more thereof. Alsoprovided is a vaccine comprising a peptide of the invention and/or apolynucleotide of the invention.

As used herein, the term “vaccine” refers to a composition comprising orencoding peptides that can be administered to a subject sensitive togluten to modulate the subject's response to gluten. The vaccine mayreduce the immunological reactivity of a subject towards gluten.Preferably, the vaccine induces tolerance to gluten.

Administration of the vaccine to a subject may induce tolerance byclonal deletion of gluten-specific effector T cell populations, forexample, gluten-specific CD4⁺ T cells, or by inactivation (anergy) ofsaid T cells such that they become less responsive, preferably,unresponsive to subsequent exposure to gluten (or peptides thereof).

Alternatively, or in addition, administration of the vaccine may modifythe cytokine secretion profile of the subject (for example, result indecreased IL-4, IL-2, TNFα and/or IFNγ, and/or increased IL-10). Thevaccine may induce suppressor T cell subpopulations, for example Tregcells, to produce IL-10 and/or TGFβ and thereby suppress gluten-specificeffector T cells.

The vaccine of the invention can be used for prophylactic treatment of asubject capable of developing sensitivity to gluten, for example,diagnosed as carrying the HLA-DQ2 and/or HLA-DQ8 gene and/or ongoingtreatment of a subject who is sensitive to gluten, for example, asubject who has celiac disease. There is considerable animal data tosupport the prophylactic activity of immunodominant peptides for variousautoimmune and model immune conditions, for example, experimentalallergic encephalitis.

As used herein, the term “treatment” includes abrogating, inhibiting,slowing, or reversing the progression of a disease or condition, orameliorating or preventing a clinical symptom of the disease (forexample, celiac disease) or condition.

The amount of vaccine (or agent, peptide, polynucleotide and/or APC) tobe administered is referred to as the “effective amount”. The term“effective amount” means the amount sufficient to provide the desiredtherapeutic or physiological effect when administered under appropriateor sufficient conditions. Single or multiple doses may be administered.Undesirable effects, for example, side effects, are sometimes manifestedalong with the desired therapeutic effect; hence, a practitionerbalances the potential benefits against the potential risks indetermining an appropriate “effective amount”. The exact amount requiredwill vary from subject to subject, depending on the species, age, sizeand general condition of the subject, mode of administration and thelike. Thus, it may not be possible to specify an exact “effectiveamount”. However, an appropriate “effective amount” in any individualcase may be determined by one of ordinary skill in the art using onlyroutine experimentation.

The vaccine (or agent, peptide, polynucleotide and/or APC) modifies theT cell response to wheat, barley and rye in the subject, and preferablywheat, barley, rye and oats, as represented by gliadin, secalin,hordein, glutenin and optionally avedin proteins. Thus, a subjecttreated according to the invention preferably is able to eat at leastwheat, rye, barley and optionally oats without a significant T cellresponse which would normally lead to symptoms of celiac disease.

The individual components of an agent of the invention may beadministered in the same composition or in different compositions or acombination thereof (for example, the first and second peptide definedherein in one composition, and the third peptide in a separatecomposition). If in different compositions, they may be administeredsimultaneously or sequentially.

The agent or vaccine may include a pharmaceutically acceptable carrier.The term “pharmaceutically acceptable carrier” refers to molecularentities and compositions that do not produce an allergic, toxic orotherwise adverse reaction when administered to a subject, particularlya mammal, and more particularly a human. The pharmaceutically acceptablecarrier may be solid or liquid. Useful examples of pharmaceuticallyacceptable carriers include, but are not limited to, diluents,excipients, solvents, surfactants, suspending agents, buffering agents,lubricating agents, adjuvants, vehicles, emulsifiers, absorbants,dispersion media, coatings, stabilizers, protective colloids, adhesives,thickeners, thixotropic agents, penetration agents, sequestering agents,isotonic and absorption delaying agents that do not affect the activityof the active agents of the invention.

The carrier can be any of those conventionally used and is limited onlyby chemico-physical considerations, such as solubility and lack ofreactivity with the active agent, and by the route of administration.Suitable carriers for this invention include those conventionally used,for example, water, saline, aqueous dextrose, lactose, Ringer'ssolution, a buffered solution, hyaluronan, glycols, starch, cellulose,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, glycerol, propylene glycol, water, ethanol, and the like.Liposomes may also be used as carriers.

Techniques for preparing pharmaceutical compositions are generally knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980.

The term “adjuvant” generally refers to an immunostimulatory substancedesigned to enhance the immunogenicity of one or more peptides definedherein. Preferably, the adjuvant does not produce a Th1 response andfurther, promotes immune tolerance and/or reduces inflammation. Suitableadjuvants include 1) an aluminium-based mineral salt adjuvant, forinstance an Al(OH)₃ gel or aluminium phosphate, but may also be a saltof calcium, iron or zinc; and 2) dexamethasone (Kang et al., 2008).

Administered may be orally, topically (percutaneous), parenterally, byinhalation spray or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers. The term“parenteral”, as used herein includes intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, subconjunctival,intracavity, transdermal and subcutaneous injection, aerosol foradministration to lungs or nasal cavity, or administration by infusionby, for example, osmotic pump.

The active compounds of the invention may be in a form suitable for oraluse, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups or elixirs. Compositions intended for oral use maybe prepared according to methods known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavouring agents, colouring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations.

Tablets

Tablets containing the active ingredient in admixture withpharmaceutically acceptable excipients may also be manufactured by knownmethods. The excipients used may be for example, (1) inert diluents suchas calcium carbonate, lactose, calcium phosphate or sodium phosphate;(2) granulating and disintegrating agents such as corn starch, oralginic acid; (3) binding agents such as starch, gelatin or acacia, and(4) lubricating agents such as magnesium stearate, stearic acid or talc.The tablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed. They may also be coated to form osmotic therapeutictablets for controlled release.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They may also be in the form of soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous Suspensions

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may include: (1) suspending agents such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; or(2) dispersing or wetting agents such as PEG esters of C₂-C₁₈ fattyacids, TWEEN® 80 or polyethylene oxide sorbitan monooleate, BRIJ® orpolyoxyethylene alcohol, TRITON™-X or Polyethylene glycolp-isooctylphenyl ether, TRITON™-N or Polyoxyethylene branchednonylcyclohexyl ether, and TRITON™ A-20 or 4-(1,1,3,3-Tetramethylbutyl)phenol, polymer with formaldehyde and oxirane, DECON, Tris or2-amino-2-hydroxymethyl-1,3-propanediol and CREMOPHOR® EL or polyoxyl 35hydrogenated castor oil.

The aqueous suspensions may also contain one or more preservatives, forexample, ethyl or n-propyl p-hydroxybenzoate; one or more colouringagents; one or more flavouring agents; and one or more sweetening agentssuch as sucrose, aspartame or saccharin.

Oily Suspensions

Oily suspension may be formulated by suspending the active ingredient ina vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, a fish oil which contains omega 3 fatty acid, or in amineral oil such as liquid paraffin. The oily suspensions may contain athickening agent, for example beeswax, hard paraffin or cetyl alcohol.Sweetening agents and flavouring agents may be added to provide apalatable oral preparation. These compositions may be preserved by theaddition of an antioxidant such as ascorbic acid.

Dispersible Powders and Granules

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They provide the active ingredient in a mixture witha dispersing or wetting agent, a suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example, those sweetening, flavouring and colouringagents described above may also be present.

Emulsion

The pharmaceutical composition(s) may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil such asolive oil or arachis oils, or a mineral oil such as liquid paraffin or amixture thereof. Suitable emulsifying agents include gum acacia, gumtragacanth, soy bean, lecithin, polyoxyethylene oxide sorbitanmonooleate (TWEEN® 80). The emulsions may also contain sweetening andflavouring agents.

Syrups and Elixirs

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol, aspartame or sucrose.Such formulations may also contain a demulcent, preservative, flavouringand colouring agents.

Injectables

The pharmaceutical composition(s) may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may be a suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable carriers that may be employed arewater, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose, any bland fixed oil may beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

Compositions suitable for parenteral administration include, but are notlimited to, aqueous and non-aqueous sterile injection solutions.Examples of appropriate delivery mechanisms for subcutaneousadministration include, but are not limited to, implants, depots,needles, capsules, and osmotic pumps.

Sustained-Release Compositions

Sustained-release compositions may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers which matrices are in the form of shaped articles,for example, films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods.

The active agent may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

Microencapsulation for sustained release has been successfully performedwith human growth hormone (rhGH), interferon (rhIFN), interleukin-2, andMN rgp120. The sustained-release formulations of these proteins weredeveloped using PLGA polymer due to its biocompatibility and wide rangeof biodegradable properties. The degradation products of PLGA, lacticand glycolic acids, can be cleared quickly within the human body.Moreover, the degradability of this polymer can be adjusted from monthsto years depending on its molecular weight and composition.

Gene Therapy

In a further embodiment, a polynucleotide encoding one or more peptidesdefined herein is inserted into a recombinant expression vector for thepurposes of administration to the subject.

The term “recombinant expression vector” refers to a plasmid, virus orother vehicle known in the art that has been manipulated by insertion orincorporation nucleic acid encoding one or peptides. Such expressionvectors contain a promoter sequence which facilitates the efficienttranscription in the host of the inserted genetic sequence. Theexpression vector typically contains an origin of replication, apromoter, as well as specific genes which allow phenotypic selection ofthe transformed cells.

In one embodiment, the viral vector is derived from adeno-associatedvirus (AAV) and comprises a constitutive or regulatable promoter capableof driving sufficient levels of expression of the peptides definedherein. Preferably, the viral vector comprises inverted terminal repeatsequences of AAV, such as those described in WO 93/24641. In a preferredembodiment, the viral vector comprises polynucleotide sequences of thepTR-UF5 plasmid. The pTR-UF5 plasmid is a modified version of thepTR.sub.BS-UF/UF1/UF2/UFB series of plasmids (Zolotukiin et al., 1996;Klein et al., 1998).

Promoters useful with the subject invention include, for example, thecytomegalovirus immediate early promoter (CMV), the human elongationfactor 1-α promoter (EF1), the small nuclear RNA promoters (U1a andU1b), α-myosin heavy chain promoter, Simian virus 40 promoter (SV40),Rous sarcoma virus promoter (RSV), adenovirus major late promoter,β-actin promoter and hybrid regulatory element comprising a CMVenhancer/β-actin promoter. These promoters have been shown to be activein a wide range of mammalian cells.

The promoters are operably linked with heterologous polynucleotideencoding one or more peptides defined herein. By “operably linked,” itis intended that the-promoter element is positioned relative to thecoding sequence to be capable of effecting expression of the codingsequence.

Also contemplated for use with the vectors of the present invention areinducible and cell type specific promoters, for example, Tet-induciblepromoters (Clontech, Palo Alto, Calif.) and VP16-LexA promoters(Nettelbeck et al., 1998).

Transcriptional enhancer elements which can function to increase levelsof transcription from a given promoter can also be included in thevector. Enhancers can generally be placed in either orientation, 3′ or5′, with respect to promoter sequences. In addition to the naturalenhancers, synthetic enhancers can be used in the present invention, forexample, a synthetic enhancer randomly assembled from Spc5-12-derivedelements including muscle-specific elements, serum response factorbinding element (SRE), myocyte-specific enhancer factor-1 (MEF-1),myocyte-specific enhancer factor-2 (MEF-2), transcription enhancerfactor-1 (TEF-1) and SP-1 (Li et al., 1999; Deshpande et al., 1997;Stewart et al., 1996; Mitchell and Tjian, 1989; Briggs et al., 1986;Pitluk et al., 1991) can be used in the vector.

The gene therapy methods can be performed by ex vivo or in vivotreatment of the patient's cells or tissues. Vectors can be introducedinto suitable cells, cell lines or tissue using methods known in theart. The viral particles and vectors can be introduced into cells ortissue in vitro or in vivo. Methods contemplated include transfection,transduction, injection and inhalation, for example, vectors can beintroduced into cells using liposomes containing the subject vectors, bydirect transfection with vectors alone, electroporation or by particlebombardment.

Dosage

It is especially advantageous to formulate the active in dosage unitform for ease of administration and uniformity of dosage. “Dosage unitform” as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active agent calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms are dictated by anddirectly dependent on the unique characteristics of the active agent andthe particular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active agent for thetreatment of subjects. Alternatively, the compositions may be presentedin multi-dose form.

Examples of dosage units include sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use.

The agent or vaccine may also be included in a container, pack, ordispenser together with instructions for administration.

The actual amount administered (or dose or dosage) and the rate andtime-course of administration will depend on the nature and severity ofthe condition being treated. Prescription of treatment, for example,decisions on dosage, timing, frequency, etc., is within theresponsibility of general practitioners or specialists (including humanmedical practitioner, veterinarian or medical scientist) and typicallytakes account of the disorder to be treated, the condition of thesubject, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of techniques and protocols canbe found in Remington's Pharmaceutical Sciences, 18th Ed. (1990), MackPublishing, Company, Easton, Pa., U.S.A.). The dose, dose frequency,duration, route of administration and need for maintenance therapy couldbe based upon the criteria for other peptide immunotherapeutics.

Effective amounts may be measured from ng/kg body weight to g/kg bodyweight per minute, hour, day, week or month.

When in vivo administration of an agent or vaccine of the invention isemployed, normal dosage amounts may vary from about 10 ng/kg to up to100 mg/kg of mammal body weight or more per day, preferably about 1μg/kg/day to 10 mg/kg/day, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature.

Toxicity and therapeutic efficacy of the agent or vaccine can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals by determining the IC₅₀ and the maximal tolerateddose. The data obtained from these cell culture assays and animalstudies can be used to formulate a range suitable for humans.

Diagnosis and Efficacy of Treatment

The peptides defined herein are also useful as a diagnostic agent.

In one example, gluten tolerance is assessed by measuring IL-10 and/orTGFβ secreted from stimulated cells, for example, Treg cells, exposed tothe peptides defined herein. Treg cells are characterised by theircapacity to produce large amounts of IL-10 and TGFβ. IL-10 is consideredto be one of the main cytokines involved in immunosuppression; a targetfor suppression seems to be the transcriptional control of IL-2 ineffector cells.

In another example, gluten tolerance is assessed by measuring IFNγsecreted from stimulated cells, for example, gluten-specific CD4⁺ Tcells.

The diagnostic test may be performed in vitro using whole blood or cellsisolated and/or fractionated therefrom.

In one example, the cells have been previously exposed to one or more ofthe peptides (either alone, conjugated to an MHC molecule or fragmentthereof, or peptide loaded APC). In another example, the cells arestimulated in vitro by coincubation with the peptides (either alone,conjugated to an MHC molecule or fragment thereof, or peptide loadedAPC).

The direct T cell mediated effects of the agent can be monitored byfunctional assays utilising cells isolated from peripheral blood ortissue (for example, the small intestine). Effects of peptideadministration down stream to cognate T cells could be assessed usingimmune cell types, tissues, biological fluids (for example, plasma,intestinal secretions, urine or stool).

In general the biological effects of peptides recognised by cognate Tcells are either pro-inflammatory or tolerogenic, depending on the doseregimen, mode of administration and whether the peptides are modified orco-administered with another compound that has immunological properties,for example, an adjuvant. These and other peptides selected for use inpeptide based therapeutic vaccines are generally short (<29 aminoacids), aqueous-soluble, without innate immune effects and recognised bya substantial proportion of pathogenic T ells. Based upon observationsin animal models of T cell mediated disease and in other human diseases,initial administration would be followed by activation of cognate Tcells. However, repeated administration of the agent is expected toinduce T cell anergy and/or tolerance. Ongoing regular peptideadministration would be expected to maintain tolerance to gluten,suppress inflammation in the small intestine and inhibitpro-inflammatory gluten-specific T cells throughout the body.

Hence, the key marker of therapeutic success would be the absence ofinflammation in the small intestine following deliberate gluteningestion. Surrogate markers of immunity likely to predict normal orinflamed intestinal tissue after gluten ingestion includes a wide rangeof assays utilizing pure or crude mixtures of immune cells, biologicalfluids, or tissue samples, to measure soluble or cell-associatedproteins or small molecules associated with immune activation,inflammation, or tolerance. These assays are well-known toimmunologists, immuno-histologists, and clinicians familiar with immunediseases in rodents, humans, and in particular, celiac disease. Markers,more specifically, that assess the activity of celiac disease andgluten-induced immunity include small bowel histology, serum IgA and IgGspecific gliadin (protein or peptide) and for various host proteinsincluding tTG.

Generic and specific markers of immunity in celiac disease that might beadapted for use in monitoring the peptide immunotherapy for celiacdisease or for diagnosis of celiac disease include the following:

(a) Direct effects of peptides on the CD4⁺ T cell isolated from blood ortissue can be monitored ex vivo/in vitro by peptide-stimulated cytokinerelease, T-cell proliferation, or determination of CD4⁺ T cell markersthat may be altered in vivo.(b) The frequency and phenotype of individual CD4⁺ T cells specific forthe peptides or gluten generally can be assessed by direct enumerationof cells, for example, by FACS analysis. Oral ingestion of gluten inpatients with celiac disease normally following a gluten free diet isknown to stimulate T cells specific for the peptides and glutengenerally. A clinical test such as gluten challenge may be used toassess the T cells induced in blood or other tissues. The phenotype ofisolated T cells could then be assessed fresh or following short-termexpansion in vitro. Assays of T cells may rely upon MHC-peptidecomplexes, antigen-stimulated intracellular cytokine, or other cellsurface markers induced on antigen-activated T cells. Functional statusof CD4⁺ T cells is correlated with the presence of various cell-surfaceand intra-cellular markers, for example, activation markers includingCD25 and CD69, or of “tolerance” and regulatory T cell function, forexample, GITR and FOXP3. Production of cytokines such as IFNγ, IL-4,IL-5 and IL-13, and of IL-17 would be considered pro-inflammatory forclassic Th1, Th2 or Th17 pro-inflammatory immune responses. In contrast,secretion of IL-10 and TGFβ are associated with tolerogenic immuneresponses. It would be expected markers of pro-inflammatory immuneresponses would decline and/or markers of tolerogenic immune responseswould strengthen.(c) Effects of peptides on CD4⁺ T cells can also be measured usingmixtures of cells, for example, whole blood, PBMC, mononuclear cellsisolated from tissue, or using tissue incubated with the peptides.Assays capable of measuring individual or multiple proteins or RNAencoding relevant immunological or disease-associated proteins such ascytokines and chemokines could be assessed after short-term incubationwith the peptides. Assays such as IFNγ ELISpot using PBMC before and orafter administration of gluten or peptides themselves to the patient, ormultiplex assays of chemokines and cytokines using PBMC are capable ofdetecting the biological effects of peptide-specific T cells frompatients. The therapeutic effect of the peptides would be indicated by ashift from markers associated with pro-inflammatory immune responses tomarkers associated with immune tolerance (for example, IL-10) andgeneral reduction in pro-inflammatory markers such as IFNγ.(d) Effects of peptides on tissue may be practical; functional assayscould take the form of direct application of peptide to the skin toassess delayed-type hypersensitivity, as in the Mantoux test fortuberculosis, which involves intradermal application of PPD (purifiedprotein derivative) and assessment of the diameter of redness at theinjection site 24-72 h later. The peptides may also be applied to othermucosal and skin sites to assess in the same manner. In clinicalpractice, it is both the peptide and grain derived protein-stimulatedimmune response that is important in celiac disease. For example, it ispredicted that immunotherapy using the selected peptides would not onlylead to suppression of the immune response stimulated by T cellsspecific for the peptides but also “tolerance” would be “infectious” andalso lead to suppression of pro-inflammatory immunity to othergluten-derived peptides and gluten itself. Hence, the effects of thepeptide therapy could also be monitored using gluten from various grains(wheat, rye, barley) in celiac disease, in place of peptide in theassays described above. Indeed, peptide therapy for cat-sensitive asthmahas been monitored by such a skin test utilizing the whole proteinantigen from which the therapeutic peptides are derived (Oldfield etal., 2002).(e) Ultimately, the clinical effects of the peptide immunotherapy wouldbe assessed by histologic examination of tissues exposed to dietarygluten, typically the small bowel, but in experimental settings oral andrectal mucosa have also bee assessed, and in principle other sites suchas oesophagus and colon might also be assessed. Tissue from these sitescould be collected by direct visualization, typically by endoscopicbiopsy. Direct visualization by endoscopy has also been used to diagnoseceliac disease according to the appearance of the mucosa—villous atrophycan be assessed by standard as well as magnifying and capsule endoscopy.Hence, the tolerogenic effects of the peptides may be assessed simply bydetection of macroscopic tissue damage in the gastrointestinal tract.(f) Immunoglobulin specific for the peptides or other gluten peptides,or autoantigens relevant to celiac disease would provide markers ofgluten immunity relevant to disease activity, and to opsonising activitythat may compromise the therapeutic effects of the peptides themselves.(g) Presence of markers associated with anaphylaxis, such as peptide- orgluten-specific IgE or histamine release by peripheral blood basophilsmay also be used to predict complications of peptide immunotherapy andneed to adjust or cease therapy.

Food Test

The invention also provides a method of determining whether acomposition or food is capable of causing celiac disease, the methodcomprising detecting the presence of the agent of the invention, thepeptide of the invention and/or the polynucleotide of the invention inthe composition or a food sample. Typically this is performed by using abinding assay in which one or more compounds which bind one or morepeptides defined herein in a specific manner is contacted with thecomposition and the formation of peptide/compound complex(es) isdetected and used to ascertain the presence of the peptide(s). In oneexample, the compound is an antibody. Any suitable format of bindingassay can be used. Typically, the assay utilises monoclonal antibodiesto gluten peptides in a non-competitive, sandwich type ELISA. Foodsamples may first be extracted, optionally diluted and then tested inthe assay.

The composition or food typically comprises material from a plant thatexpresses gluten. Such material may be a plant part, such as a harvestedproduct (for example, seed). The material may be processed products ofthe plant material, such as a flour or food that comprises gluten. Theprocessing of food material and testing in suitable binding assays isroutine (see for example, Kricka, 1998). The composition or foodmaterial may be treated with tTG prior to being contacted with thecompound.

In one embodiment, the composition or food material is contacted with atleast 2, 3, 5, 10 or more antibodies which are specific for peptidesdefined herein in deamidated and/or non-deamidated form. Preferably, theantibodies are directed against sequences that are protease resistantand allow for the detection of α, β, γ and ω gliadins, and LMW and HMWglutenins in wheat, B, C and D hordeins in barley, β, γ and ω secalinsin rye, and optionally avenins in oats.

Antibodies directed against the peptides/epitopes defined herein may beprovided in kit form for use in an assay for the detection and/orquantification of gluten in foods.

Protease Identification

The present invention also provides a method of identifying a proteasethat can cleave a peptide as defined herein, the method comprisingcontacting the peptide with a protease under conditions to effectspecific cleavage of the peptide to produce a proteolytic product anddetecting the proteolytic product produced. In one example, theproteolytic product is detected, for example, using SDS-PAGE, HPLC,ELIZA, or Western Blot. In a further example, the peptide is fused to afluorescent donor and a quenching acceptor so as to enableintramolecular resonance energy transfer between the fluorescent donorand the quenching acceptor. Upon cleavage, the donor and acceptor areseparated, allowing detection of the donor's fluorescent emission.Typically the peptide separates the fluorescent donor and the quenchingacceptor at a distance of less than about 100 angstroms. The fluorescentdonor can be attached to the peptide's C-terminus, and the quenchingacceptor can be attached to the peptide's N-terminus, or vice versa.

EXAMPLES Example 1: Determination of Immunodominant Peptides Subjects

Volunteers were adults aged 18-70 years and following strict gluten freediet. All volunteers possessed genes encoding both HLA DQAB1*05 and HLADQB1*02 as determined by PCR with sequence-specific primer mixes ofperipheral blood DNA (Bunce et al., 1995; Olerup et al., 1993; Mullighanet al., 1997). Volunteers with celiac disease were diagnosed on thebasis of ESPGAN criteria (Report of Working Group of European Society ofPaediatric Gastroenterology and Nutrition, 1990). Subjects with celiacdisease undergoing gluten challenge were on gluten free diet for atleast 1 month and claimed to be compliant (positive tTG-IgA or EMA wasan exclusion). Healthy HLA DQ2 subjects (endosmysial IgA negative) hadfollowed a strict gluten free diet for 4 weeks before commencing glutenchallenge.

Three-Day Gluten Challenge

Wheat Challenge:

Two 50 g slices for breakfast and for lunch of either Sainsbury's“standard white sandwich bread” (UK—to assess the Pilot library), orotherwise Baker's Delight “white bread block loaf”).

Barley Challenge:

Pearl barley (Ward McKenzie, Altona, Australia) cooked as risotto (150 gdry weight daily). Risotto servings were divided into equal servings forbreakfast, lunch and dinner.

Rye Challenge:

Daily consumption of 100 g dry weight rye flour in the form of muffinseaten throughout the course of the day beginning at breakfast. Ryeflour-sourced was either from rye grown in “isolation” at Long AshtonResearch Station, UK and subsequently hand milled (for assessment of thePilot library), or from Biodynamic rye flour (Eden Valley BiodynamicFarm, Dumbleyung, Australia).

Combined Wheat, Barley and Rye Challenge:

Two muffins consisting of 25 g wheat flour (White Wings, GoodmanFielder, Australia), 22 g barley flour (Four Leaf Milling, Tarlee, SouthAustralia), and 22 g rye flour (Four Leaf Milling, Tarlee, SouthAustralia) were eaten each day.

Antigens

Synthetic peptides (purity>70%) were purchased from Research Genetics(USA), Mimotopes (Australia), or Pepscan (Netherlands). Deamidation withguinea pig liver tTG (Sigma T5398) was as described previously (Andersonet al., 2000). Peptides (2 mg/ml) or gliadin (Sigma G3375) wereincubated for 4 hours, 37° C. in 10-fold excess with chymotrypsin (SigmaC3142) or trypsin (Sigma T1426) in ammonium bicarbonate (pH 8), or withpepsin (Sigma P6887) in 5% acetic acid (pH 2.5), then neutralised to pH7 with NaOH, and finally boiled for 15 minutes. Prolamin proteinconcentrations were determined by BCA method (Pierce, USA). Hordein andsecalin fractions were prepared from rye and barley grown in isolationfrom other grains, hand-milled flour, and fractionated according topublished methods (Tatham, A. S., Gilbert, S. M., Fido R. J., andShewry, R. Extraction, separation, and purification of wheat glutenproteins and related proteins of barley, rye, and oats. In: Marsh M,ed., Celiac disease methods and protocols. Totowa: Humana (2000) pp55-73).

Peptide Libraries

Wheat, barley and rye gluten peptide libraries were designed byalignment and phylogeny (“Pilot” library, see Sequence Listing, Tables 3and 4, or using a customised algorithm applied to entries for gliadins,glutenins, hordeins and secalins in NCBI Genbank at 2006 in theirgenome-encoded (wildtype) sequence (“Comprehensive” library), or bothwildtype and in silico tTG deamidated sequence (“Verification” library)according to defined deamidation motifs (Beissbarth, et al., 2005).

TABLE 3 Gluten peptide libraries. Library Pilot ComprehensiveVerification Aim Feasability of comprehensive1. Define hierarchy and identity of T cell Define the range of peptidesT cell eptiope mapping in celiac  stimulatory wheat, barley, rye and oatin gluten recognised by disease gluten peptides; andceliac donor-derived 2. Define lead compound for peptide-intestinal and peripheral based therapeutic vaccine for HLA DQ2⁺blood T cell clones specific celiac disease for immunodominant epitopesUse PBMCs in (polyclonal) T cell IFNγSame as Pilot, but scaled up to confirm T cell clones from peripheralELISpot assay drawn day0 vs. day-6 ofPilot data and test all gluten proteins fromblood and intestinal biopsies gluten challenge of HLA DQ2⁺ celiac vs.wheat, rye, and barley. healthy UK donors Genbank September 2001June 2003 October 2006 DatabaseSpecies: T. aestivum (wheat), T. aestivumSpecies: T. aestivum (wheat), H. vulgare Species: T. aestivum (wheat),Search subsp. (barley), S. cerale (rye) T. aestivum subsp., H.Terms: alpha-gliadin, beta-gliadin,Terms: gluten, gliadin, glutenin, hordein, vulgare (barley), S. ceralegamma-gliadin, omega-gliadin secalin (rye) Terms: gliadin, hordein,secalin Search 61 α/β-, 47 γ-, 3 ω-gliadins53 α/β-, 53 γ-, 2 ω-gliadins, 77 LMW, 55 58 α/β-, 48 γ-, 5ω-gliadinsresults HMW glutenins, 59 hordeins, 14 secalins 86 hordeins, 16 secalinsDesign All wild-type 12 mers All wild-type 12 mersAll wild-type and tTG- Alignment by phylogeny Library algorithmdeamidated 10 mers: gliadin, Meg aAlign ClustalW hordein, and secalinSize 652 20 mers encompassing 3997 12 mers 20 mers (12 mers, 9 mers)18 mers (10 mers, 9 mers) and 3372 9 mers Gliadin: 721 (4465, 3739)Gliadin: 1363 (8114, 7561) LMW glutenin: 645 (3945, 3164)Hordein: 1338 (8557, 8117) HMW glutenin: 786 (4799, 3630)Secalin: 327 (2105, 1955) Hordein: 416 (2672, 2413)Secalin: 155 (957, 811) Termini H-, -OH (free) H-, -OH (free)H-, -OH (free) Amount 0.6 μmol 1 μmol 4× 0.5 μmol: gliadin,hordein, secalin QA 2 standards per 96 20 mers2 standards per 96 20 mers All assessed by LC-MS.Amino acid analysis and HPLC: Amino acid analysis and HPLC:1320/1363 gliadin 18 mers, IKDFHVYFRESRDALWKGPG (SEQIKDFHVYFRESRDALWKGPG (SEQ 1311/1338 hordein 18 mers, ID NO: 519)ID NO: 519) and 321/327 secalin 18 mers.Purity 50, 41-56% (median, range, n = 7)Purity 64, 55-71% (median, range, n = 31) 10 mer sequences in 27VLQQHNIAHGSSQVLQESTY (SEQ One 20 mer from each 96 blockhordein and 6 secalin 18 mers ID NO: 520) Purity 36, range: 5-68% (n =31) with incorrect mass were Purity 17, 16-23% (n = 7)synthesised as 108 12 mers (all with correct mass) DissolutionACN 10% 0.1 M HEPES 50% Aqueous acetonitrile 50% Aqueous acetonitrileStock 10 mg/ml 50 mg/mL 25 mg/mL

ELISpot Assay

IFNγ ELISpot assays (Mabtech, Sweden) using 96 well plates (MSIP-S45-10;Millipore, Bedford, Mass.) were performed using Peripheral bloodmononuclear cells (PBMC) from blood drawn between 0800 hours and middayon the sixth day after commencing gluten challenge as previouslydescribed. Briefly, ELISpot plates were coated with sterile captureanti-cytokine antibody at 1:100 concentration (50 μl/well) diluted inPBS and wrapped in foil overnight at 4° C. Prior to use, each plate waswashed three times with sterile PBS and non-specific binding blocked byaddition of RPMI with 10% FCS (50 μl/well) for 2 hours at 37° C. Antigenat 5× concentration was added to each well (25 μl) followed by additionof freshly isolated PBMC suspended in complete medium (100 μl) andincubated overnight (16-20 hours) at 37° C. in a 5% CO₂ incubator. Cellsand culture medium were then discarded and the plate washed once withcold distilled water then three times with PBS with 0.05% TWEEN®-20(Sigma P2287, St Louis, USA) and three times in PBS (200 μl/well eachwash). Biotinylated anti-cytokine mAb (1:1000) diluted in PBS with 0.5%FCS (50 μl/well) was incubated for 2 hours at room temperature. Wellswere washed six times with PBS (200 μl/well), and Streptavidin-ALP(1:1000) added (50 μl/well) and incubated for 1 hour at roomtemperature. After washing, BCIP-NBT developer substrate was added (50μl/well) and spots allowed to develop. Developing was terminated bywashing under cold water when spots were first visible. The number ofspot-forming units (SFU) in individual wells was enumerated withcomputer-assisted video image analysis (AID ELISpot Reader System, AIDAutoimmun Diagnostika GmbH, Strassberg, Germany). Mycobacteriumtuberculosis purified protein derivative (PPD RT49) (5 μg/ml) and/ortetanus toxoid (CSL) (10 light forming units/ml) were positive controlantigens.

Isolation of T Cell Clones

PBMC were isolated from heparinised whole blood using Ficoll-Paque Plusin Leucosep tubes. Lamina propria mononuclear cells (LPMC) were isolatedfrom small intestinal biopsies by first treating samples with 1 mM DTTin PBS, followed by two incubations at 37° C. for 30 minutes in 2.4 U/mLDispase II. Biopsies were then minced and incubated at 37° C. for 1 hourin 2 U/mL Liberease Blendzyme 3 and RPMI. PBMC and LPMC were washedthree times in PBS. Typically, between 0.5 and 1×10⁶ LPMC were recoveredand were mixed with 1.5-3 million autologous PBMC irradiated at 2000rads.

PBMC and LPMC were stained with 0.1 μM CFSE and were plated out in 96well plates at 2×10⁵ cells/well, as previously described (Mannering etal., 2003; Mannering et al., 2005). Peptide and protein antigens wereused at 32 μg/mL and 100 μg/mL respectively. Between 7 and 10 dayslater, CD4⁺ proliferation was measured by flow cytometry (FACSAria, BD).CD4⁺ CFSE^(dim) PI− cells were sorted into a single well of a 96 wellplate containing 2×10⁵ PBMC (irradiated at 2000 rads), 2×10⁴ JY-EBV(irradiated at 5000 rads), 20 U/mL recombinant human IL-2, 5 ng/mLrecombinant human IL-4 and 30 ng/mL anti-CD3 (OKT3) in media. Cells werefed every 7 days for 2 weeks with media containing cytokines to give afinal concentration of 20 U/mL IL-2 and 5 ng/mL IL-4. On day 25, growingclones were identified and expanded into 48 well plates in mediaincluding 20 U/mL IL-2 and 5 ng/mL IL-4. Antigen specificity wasdetermined by ³H-thymidine proliferation assay or IFNγ ELISpot. Largescale expansion of specific clones was carried out in culture flaskscontaining 30 ng/mL OKT3 in 15 ml media with 5×10⁷ PBMC (irradiated 2000rads) and 5×10⁶ JY-EBV (irradiated 5000 rads). After 24 hours, IL-2 wasadded to a final concentration of 50 U/mL. On day-3, the expansion waswashed and resuspended in 25 ml of media containing 50 U/mL IL-2. Onday-7, the cells were split in half and topped up with 12.5 ml of mediacontaining IL-2 at a final concentration of 50 U/mL. Expanded cells wereexamined for antigen specificity on day-10 by ³H-thymidine proliferationassay or IFNγ ELISpot.

Characterisation of T Cell Clones

Expanded antigen-specific clones were tested for clonality using theIOTest Beta Mark (Beckman Coulter). Negative clones were confirmed asclonal by PCR of the TCR Vβ chains. HLA-restriction was determined byanti-HLA-DR (10 μg/ml Clone L243) and HLA-DQ (10 μg/ml Clone SPVL3)antibodies. Secretion of IFNγ, IL-4, IL-5, IL-10, IL-13, and IL-17 byclones to cognate antigen was determined in ELISpot assays utilisingirradiated APCs (2000 rads) from HLA DQ2⁺ HLA DQ8⁻ donors. Lysine scansof SEQ ID NOs:228, 229, and 230 (NPL001, NPL002, and NPL003,respectively) were carried out in ELISpot or proliferation assays usingclones specific for these peptides.

Data Analysis

ELISpot responses were considered significant when SFU were both greaterthan four times medium alone and greater than 10 SFU/well. Proliferationassays were considered significant when stimulation indices (SI) weregreater than 3. Data sets were normalised for inter-donor or inter-clonevariability by expressing SFU or SI as a percentage of the most reactivepeptide, peptide pool, or cocktail tested. Reactive peptides and peptidepools were assigned a “score” between 0 and 100, equal to the meannormalised response of donors who responded to at least one peptide orpool.

Example 2: Determination of the Primary Dominant Peptides Using FreshPolyclonal T Cells Induced by In Vivo Gluten Challenge

In previous studies, it has been found that gluten-specific T cells areat their peak in blood 6 days after HLA DQ2⁺ celiac disease donorscommence oral gluten challenge. On day-6, IFNγ ELISpot responses of PBMCfrom celiac disease donors to optimal concentrations of tTG-treatedgliadin (500 μg/ml) and α-gliadin p57-73 QE65 (SEQ ID NO:8) encompassingDQ2-α-I (SEQ ID NO:3) and DQ2-α-II (SEQ ID NO:4) epitopes weresignificantly correlated (r=0.80, p<0.0001). Median IFNγ ELISpotresponses to 17 mer were 51% (n=17, range: 0-155%) of those totTG-treated gliadin (500 μg/ml). However, the α-gliadin p57-73 QE65 (SEQID NO:8) was not always immuno-dominant. IFNγ ELISpot responses wereequivalent to less than 5% of those to tTG-treated gliadin in 3/17donors (Anderson et al., 2005).

Based upon these observations, it is clear that gluten peptidesadditional to the α-gliadin p57-73 QE65 (SEQ ID NO:8) and peptidesincluding the epitopes SEQ ID NOs:4 and/or 5 must also stimulate asubstantial population of T cells induced by in vivo gluten challenge.The inventors were not confident that a peptide-based immunotherapyutilising α-gliadin p57-73 QE65 (SEQ ID NO:8) and peptides including theepitopes SEQ ID NOs:4 and/or 5 would alone consistently target asufficiently large proportion of the disease-relevant gluten-specific Tcell population. The inventors hypothesised that either α-gliadin p57-73QE65 (SEQ ID NO:8) and peptides including the epitopes SEQ ID NOs:4and/or 5 were partial agonists and that sequences related to SEQ IDNOs:8, 4 and/or 5 would stimulate substantially more T cells, or thatadditional peptides encompassing immuno-dominant epitopes are presentamongst gluten proteins expressed by wheat, barley or rye.

Homology Searches

Almost all substitutions to the core five amino acids, PELPY (SEQ IDNO:22) of α-gliadin p57-73 QE65 (SEQ ID NO:8) abolish its recognition byperipheral blood T cells induced by gluten challenge.

SwissProt and Trembl databases were searched for cereal genes encoding17 mers with the sequence PELPY (SEQ ID NO:22) the equivalent wild-typesequence, PQLPY (SEQ ID NO:23). Thirteen wheat α-gliadin 17 mers werefound with PQLPY and one with PQLSY (SEQ ID NO:24) at positions 8-12,but none had the sequence PELPY. With reference to FIG. 1, ELISpotresponses are shown from a variety of 17 mers with T cell epitopesDQ2-α-I (SEQ ID NO:3), DQ2-α-II (SEQ ID NO:4), and DQ2-α-III (SEQ IDNO:5), which derive from a highly polymorphic region of the α-gliadinfamily of proteins. Normalised IFNγ ELISpot responses of PBMC from 8celiac disease donors (6 days after commencing wheat gluten challenge)to fourteen naturally occurring α-gliadin 17 mers, each of which includethe core sequence PQLPY (SEQ ID NO:23) or PQLSY (SEQ ID NO:24) are shownin FIG. 1. 17 mers are assessed with or without pre-treatment with tTGor when glutamine at position 9 (Q9) is replaced by glutamate (E9). Datarepresent mean±SEM of donor ELISpot responses normalised against that toα-gliadin p57-73 QE65 (25 μg/ml).

Two 17 mers that differed from α-gliadin p57-73 QE65 (SEQ ID NO:8) onlyby having serine substituted for proline or leucine at the C-terminalwere as active as SEQ ID NO:8 when pretreated with tTG or when glutaminewas substituted for glutamate at position 9. 17 mers including bothDQ2-α-II (SEQ ID NO:4), and either DQ2-α-I (SEQ ID NO:3) or DQ2-α-III(SEQ ID NO:5) stimulate greatest numbers of T cells. These findings werein agreement with those reported by Arentz-Hansen et al., 2000 in whicha panel of intestinal T cell clones recognised five of elevenstructurally distinct recombinant α-gliadins, but only those thatincluded DQ2-α-I (SEQ ID NO:3), DQ2-α-II (SEQ ID NO:4) or DQ2-α-III (SEQID NO:5). Several other deamidated polymorphisms of α-gliadin p57-73were weakly active and one that was not among those studied byArentz-Hansen et al., 2000, PQPQPFLPQLPYPQPQS (SEQ ID NO:25; W09), wasalmost as active as 17 mers encompassing DQ2-α-II (SEQ ID NO:4) andDQ2-α-III (SEQ ID NO:5) when pre-treated with tTG or with glutamate atposition 9, PQPQPFLPELPYPQPQS (SEQ ID NO:26). Based upon a previoussubstitution scan of α-gliadin p57-73 QE65, the inventors undertook amore permissive search for homologues with a core sequence PQ[ILMP][PST](SEQ ID NO:27) (Anderson et al., 2006)

Twelve gliadin, glutenin, hordein and secalin sequences were synthesisedbut only one, the ω-gliadin peptide, AAG17702 (141-157) was more activethan medium alone. This ω-gliadin peptide, PQQPFPQPQLPFPQQSE (SEQ IDNO:28; AAD17702 (141-157)) was 32±6% as active as α-gliadin p57-73 QE65when pre-treated with tTG or with glutamate at position 9,PQQPFPQPELPFPQQSE (SEQ ID NO:29) (25 μg/ml; mean±SEM, n=5 donors).

Epitopes for Intestinal Clones and In Vivo Gluten-Induced PeripheralBlood Polyclonal T Cells

The inventors then assessed deamidated 15 mers encompassing epitopesreported for intestinal T cell clones: GLIA-20 PFRPQQPYPQ (SEQ ID NO:30)in its deamidated form PFRPEQPYPQ (SEQ ID NO:31), DQ2-γ-I PQQSFPQQQ (SEQID NO:32) in its deamidated form PQQSFPEQE (SEQ ID NO:33), DQ2-γ-IIIQPQQPAQL (SEQ ID NO:34) in its deamidated form IQPEQPAQL (SEQ IDNO:35), DQ2-γ-III QQPQQPYP{right arrow over (Q)} (SEQ ID NO:36) in itsdeamidated form EQPEQPYPE (SEQ ID NO:37), DQ2-γ-IV SQPQQQFPQ (SEQ IDNO:38) in its deamidated form SQPEQEFPQ (SEQ ID NO:39), Glu 5 QIPQQPQQF(SEQ ID NO:40) in its deamidated form QIPEQPQQF (SEQ ID NO:41), andGlt-156 PFSQQQQSPF (SEQ ID NO:42) in its deamidated form PFSEQQESPF (SEQID NO:43), and also DQ2-γ-V LQPQQPFPQQPQQPYPQQPQ (SEQ ID NO:44), andα-gliadin p31-49 LGQQQPFPPQQPYPQPQPF (SEQ ID NO:45) (over the range0.1-100 μg/ml). In 8/9 HLA DQ2 celiac disease donors, IFNγ ELISpotresponses to deamidated gliadin were detected (median 23, range: 13-153SFU/million PBMC). FIG. 2 shows 7 donors responded to the variant ofdeamidated α-gliadin p57-73 QE65 with leucine at position 17QLQPFPQPELPYPQPQL (SEQ ID NO:46) encompassing DQ2-α-I (SEQ ID NO:3) andDQ2-α-II (SEQ ID NO:4) (5 μM) and a 33 merLQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:2; deamidated α2-gliadin56-88) (5 μM) encompassing overlapping tandem repeats of DQ2-α-I (SEQ IDNO:3) and DQ2-α-II (SEQ ID NO:4), and DQ2-α-III (SEQ ID NO:5). At anoptimal concentration (50 μM), the difference between IFNγ ELISpotresponses stimulated by the 17 mer and 33 mer were not significant. Onedonor responded to the 15 mer encompassing deamidated DQ2-γ-IV (SEQ IDNO:39), but none of the other nine epitopes were recognised by PBMCcollected on day 6 after wheat gluten challenge.

The inventors concluded that, in most individuals with HLA DQ2⁺ celiacdisease, peptides encompassing DQ2-α-I (SEQ ID NO:3), DQ2-α-II (SEQ IDNO:4) or the related DQ2-α-III (SEQ ID NO:5) epitope make a substantialcontribution to the T-cell stimulatory activity of gluten in vivo, butmany other published gluten epitopes make little if any consistentcontribution to the peptides recognised by CD4⁺ T cells induced in bloodafter gluten exposure in vivo. Conversely, other sequences that mighthave potent T stimulatory activity may have been overlooked because onlya minority of gluten proteins have been systematically assessed infunctional assays. A new approach was needed to comprehensively assesscandidate T cell epitopes in gluten from wheat, rye and barley for theircontribution to the gluten-specific T cell response associated withceliac disease.

Comprehensive Triticum aestivum Gliadin Peptide Library

In 2001, there were 111 entries in Genbank for T. aestivum α-, γ-, andω-gliadin proteins. Traditional approaches to CD4⁺ T-cell epitopemapping with 15-20 mer peptides overlapping by 10-12 amino acidsspanning each polypeptide would have produced impractically largelibraries to synthesise and screen. But phylogeny analysis and alignmentof gliadin sequences by ClustalW indicate substantial sequencesimilarities within and between each phylogenetic sub-family of gliadins(Anderson, 1991). Alignment of polypeptides and systematic but notcomputer-assisted design indicated that a 652-member library of 20 mersoverlapping by 12 amino acids would be sufficient to encompass theunique 12 mers in 111 gliadins entries then present in Genbank (seeTable 3). Divided into 83 pools of up to 8 peptides with and withoutpre-treatment by tTG, this library was practical to screen before and onday-6 after gluten challenge (one well for each pool) using PBMC from100 ml blood in overnight IFNγ ELISpot assays. A further collection of100 ml blood on day-7 could then be used to verify findings and assessindividual peptides in positive pools.

Disease Specificity of T Cell Responses to Gliadin Peptide Pools

In the initial study, the pilot gliadin library was assessed usingovernight ELISpot assays to measure the frequencies of IFNγ-secreting Tcells in blood from HLA-DQ2⁺DQ8⁻ celiac disease donors on long-termgluten free diet (GFD) (n=9) and also healthy HLA-DQ2⁺DQ8⁻ volunteers(n=9) on GFD for 4 weeks, long enough for gluten challenge to be able toinduce peripheral blood T cells in celiac volunteers (Anderson et al.,2005). Amongst the healthy donors, increases in responses to three of 83pools reached statistical significance following gluten challenge(p<0.05, Wilcoxon paired rank sum), but were inconsistent, weak andunaffected by deamidation (see FIG. 3).

Amongst the nine celiac subjects there were 7 “responders” who, on day-6after commencing gluten challenge, had at least one peptide pool thatstimulated a response more than 10 SFU/well and more than four timesthat elicited by medium alone (“background”). Comparing SFU on day-6with day-0 in the 9 celiac disease donors, there was significantinduction (p<0.05, one tail Wilcoxon paired rank sum) of T cellsspecific for 34 pools including one (pool 20) that was also weaklyrecognised by healthy donors after gluten challenge. Amongst celiacdisease donors, tTG pre-treatment increased (p<0.05, one tail Wilcoxonpaired rank sum) the frequency of peripheral blood T cells thatrecognised chymotrypsin pre-treated gliadin and also 11 of the peptidepools tested.

In order to define a hierarchy based upon the consistency and relativecontribution of pools (or in later experiments, peptides) to the overallgliadin-specific T cell population, a “score” between 0 and 100 wascalculated according to the average of “responder's”-IFNγ ELISpot(SFU/well) responses above “background” on day-6 or day-7 expressed as apercentage of their maximal response to any pool (or library peptide).

From the total of 83 tTG-treated pools, 18 (22%) had a “score” over 10on day-6 and all were associated with significant induction of responsesbetween day-0 and day-6, while 5/9 and 7/12 pools scoring between 5 and10 or between 1 and 5, respectively, on day-6 were associated withsignificant induction of responses between day-0 and day 6. Six otherpools were associated with significant induction of responses but hadscores less than 1. During subsequent analysis of peptide libraries, a“score” of 5 or greater for pools or peptides was set as an arbitrarycut-off value for T cell responses to be considered “positive” andwarranting further mapping.

It was also apparent from this initial experience that utilizing poolsof gliadin peptides was relatively inefficient as almost one quarter ofpools were positive and required deconvolution. In subsequentexperiments, individual peptides rather than pools were assessed. Toenable as many peptides to be screened as possible using PBMC from asingle 300 ml blood collection, all peptides were tTG-treated (as tTGtreatment was never associated with reduction in ELISpot responses) andlibraries were screened only on day-6 or day-0.

In 4/7 “responders”, α-gliadin pools 10 or 12 with 20 mers encompassingDQ2-α-I (SEQ ID NO:3), DQ2-α-II (SEQ ID NO:4), and/or DQ2-α-III (SEQ IDNO:5) epitopes were the most active, and in the other 3 responders,ω-gliadin pool 81 was the most active. Overall, α-gliadin pool 12 hadthe highest score (78) and next was ω-gliadin pool 81 (72). IndividualtTG-treated peptides from pools 7-13, 42-53, 68, and 78-82 were assessedwith PBMC collected from 5/7 responders on day-7 (see FIG. 4).

In all cases, several peptides from each pool were reactive. Peptidesencompassing DQ2-α-II (SEQ ID NO:4) and DQ2-α-I (SEQ ID NO:3) and/orDQ2-α-III (SEQ ID NO:5) epitopes were confirmed as the five most activein the gliadin 20 mer library, but four ω-gliadin 20 mers from pools 80and 81 were 53-65% as active as the most active α-gliadin 20 mer. Allfour of the co-gliadin 20 mers included sequences homologous to DQ2-α-I(SEQ ID NO:3) and/or DQ2-α-II (SEQ ID NO:4): namely, QPFPQPQQPFPW (SEQID NO:47; W03; B01), PFPQPQQPIPV (SEQ ID NO:48; W04), QPFPQPQLPFPQ (SEQID NO:49; W06) encompassed in SEQ ID NO:28, and three included sequencesreported to be recognised by DQ2-γ-VII epitope QQPQQPFPQ (SEQ ID NO:50)when deamidated to EQPEQPFPQ (SEQ ID NO:51) specific intestinal T cellclones.

FIG. 5 shows IFNγ ELISpot responses of PBMC from celiac disease donorsafter wheat challenge to fine map the immunogenic region of deamidatedPQQPQQPQQPFPQPQQPFPWQP (SEQ ID NO:52) (as previously described in WO2005/105129). Tissue transglutaminase-treated 15 mers spanning SEQ IDNO:52 are expressed as a percentage of the most active 15 mer for eachdonor (mean+SEM, n=8) (A). The T-cell stimulatory activity of SEQ IDNO:52 could be almost completely attributed to the deamidated sequenceencompassing homologues of DQ2-α-I (SEQ ID NO:3) and DQ2-α-II (SEQ IDNO:4), QPFPQPQQPFPW (SEQ ID NO:47). FIG. 5B shows IFNγ ELISpot responsesof PBMC from celiac disease donors after wheat challenge normalisedagainst maximal individual donor responses to the Q3 E10 variant(mean+SEM, n=6). Deamidation of Q10 in QPQQPFPQPQQPFPWQP (SEQ ID NO:53)to QPQQPFPQPEQPFPWQP (SEQ ID NO:54) is sufficient to convey optimalimmunogenicity and the double deamidated sequence, QPEQPFPQPEQPFPWQP(SEQ ID NO:55; W03-E7), is equivalent in bioactivity. FIG. 5C shows IFNγELISpot responses of PBMC from celiac disease donors after wheat (n=7),barley (n=9), or rye challenge (n=10) normalised against the most activelysine-substituted 15 mer for individual donors (mean+SEM).Lysine-substitution of the central PQPEQPF sequence (SEQ ID NO:272) ofNPL002: pyroEQPFPQPEQPFPWQP-amide (SEQ ID NO:229) (32 μg/ml) abolishedthe bioactivity of this peptide. Preincubation of PBMC from HLA DQA1*05DQB1*02 homozygotes and heterozygotes with anti-HLA-DQ but not -DRabolished overnight IFNγ ELISpot responses to this peptide (data notshown).

The peptide hierarchy observed in the initial experiment was verified byseparately assessing all 652 individual 20 mers in the Pilot Gliadinlibrary using PBMC collected 6 days after wheat challenge from 13further HLA-DQ2⁺8⁻ donors (see FIG. 4). Again there was no cleardifference in activity between 20 mers including DQ2-α-II (SEQ ID NO:4)and DQ2-α-I (SEQ ID NO:3) and/or DQ2-α-III (SEQ ID NO:5), suggestingthat fresh polyclonal T cells are rarely specific for DQ2-α-I (SEQ IDNO:3) but not DQ2-α-III (SEQ ID NO:5) or vice versa.

PBMC from 6 HLA-DQ2⁺8⁺ celiac disease donors on day-6 after commencingwheat challenge were screened against each of the 652 individual 20 mersin the Pilot gliadin library (see FIG. 4). The α-gliadin peptides thatwere most active in HLA-DQ2⁺8⁻ celiac disease donors were also the mostactive in 4 HLA-DQ2⁺8⁺ celiac disease donors after gluten challenge.

PBMC from 6 HLA-DQ2⁺8⁺ celiac disease donors on day-6 after commencing3-day challenge with pure rye were screened against each of the 652individual 20 mers in the Pilot gliadin library (see FIG. 4). Thehierarchy of T-cell stimulatory gliadin 20 mers was strikingly differentfrom that observed after challenge with wheat (see FIG. 4). T cellsmeasured by the overnight IFNγ ELISpot assay in blood after ryechallenge rarely recognised 20 mers including DQ2-α-I (SEQ ID NO:3),DQ2-α-II (SEQ ID NO:4), or DQ2-α-III (SEQ ID NO:5) epitopes. Instead theω-gliadin 20 mers including QPFPQPQQPFPW (SEQ ID NO:47) and QPFPQPQQPIPV(SEQ ID NO:48) were immunodominant.

This observation suggested that although T cell clones raised againstdeamidated wheat gluten or gliadin in vitro may often be promiscuous intheir recognition of immunodominant gliadin peptides as reported byVader et al., 2003, fresh polyclonal T cells induced by in vivo glutenchallenge do discriminate between closely related sequences. Hence, theconclusion by Vader et al., 2003 that the T cell stimulatory activity ofhordeins and secalins from barley and rye was substantially attributableto the deamidated variants of sequences PFPQPQQPF (SEQ ID NO:9) andPQPQQPFPQ (SEQ ID NO:11) being homologues of DQ2-α-I (SEQ ID NO:3) andDQ2-α-II (SEQ ID NO:4) was not confirmed using fresh PBMC from celiacdisease donors after in vivo challenge with rye. Furthermore, it wasapparent that a substantial proportion of T cells specific for thedominant sequences QPFPQPQQPFPW (SEQ ID NO:47) and PFPQPQQPIPV (SEQ IDNO:48) induced by rye challenge in vivo did not recognise DQ2-α-I (SEQID NO:3), DQ2-α-II (SEQ ID NO:4), or DQ2-α-III (SEQ ID NO:5) epitopes.

Furthermore, when compared to T cells specific for the immunodominant α-and ω-gliadin peptides, T cells specific for many epitopes reported forgliadin-specific T cell clones make little or no contribution to theoverall gliadin-specific T cell population present in blood on day-6 ofwheat challenge (see FIG. 2). Therefore, the inventors concluded thatthe immunodominance and relevance of gluten epitopes previously reportedfor intestinal T cell lines and clones in vitro frequently diverges fromthat measured by an overnight assay of polyclonal T cells in bloodfreshly isolated from celiac disease donors after in vivo glutenchallenge.

Next the inventors sought to confirm and extend the hierarchy of T-cellstimulatory peptides to all gluten proteins from bread-making wheat (T.aestivum), barley and rye in HLA-DQ2⁺8⁻ celiac disease donors. To dealwith the increasing number of gluten proteins in the NCBI Genbank and todesign peptide libraries for LMW glutenins, HMW glutenins, hordeins andsecalins, the inventors developed a novel algorithm to design customisedlibraries of minimal size to accommodate all unique sequences of, forexample, 12 mers within longer peptides, for example, 20 mers.Beissbarth, T., et al., 2005. 20 mer libraries encompassing all unique12 mers allowed wheat gluten to be assessed with PBMC from two 300 mlblood samples, and hordeins and secalins each with a single 300 ml bloodcollection. Comprehensive 20 mer libraries were designed and synthesisedas screening grade Pepsets (see Table 3), encompassing all unique 12mers in Genbank polypeptide entries present in June 2003 for gliadins(108 entries, 721 20 mers encompassing 4465 unique 12 mer candidateepitopes), LMW glutenins (77 entries, 645 20 mers, 3945 12 mercandidates) and HMW glutenins (55 entries, 786 20 mers, 4799 12 mercandidates) of T. aestivum, hordeins of H. vulgare (59 entries, 416 20mers, 2672 12 mer candidates), and secalins of S. cereale (14 entries,155 20 mers, 957 12 mer candidates).

PBMC from HLA-DQ2⁺8⁻ celiac disease donors collected on day-6 aftercommencing 3-day wheat challenge were used to screen the tTG-treatedgliadin library and half the LMW glutenin library (n=20), and the secondhalf of the LMW glutenin library and HMW glutenin library (n=26). PBMCfrom 21 celiac disease donors 6 days after commencing barley challengewere used to screen the hordein library, and PBMC from 19 further donors6-days after commencing rye challenge were used to screen the secalinlibrary. IFNγ ELISpot responses to tTG-treated Pepset library peptideswere above background levels in 27/46 donors after wheat challenge, in12/21 after barley challenge and 8/19 after rye challenge.

To facilitate selection of 20 mers for fine mapping in “second round”libraries, the inventors adapted an expectation maximization (EM)approach used for analysis of microarray data (Beissbarth et al. (2005).All individual donor datasets were analysed by the EM algorithm toderive the variables λ and p to describe the IFNγ ELISpot response toeach 20 mer. The variable λ describes the relative strength of theELISpot response, and the variable p describes the proportion of donorsresponding. Each first round library 20 mer was fine mapped in secondround libraries if the product of λp was at least 5% of the most activefirst round library peptide for each grain.

Second round libraries were designed by reducing selected 20 mers to 9overlapping 12 mers. If any 12 mer incorporated glutamine at position 7and it conformed to the deamidation motif defined for tTG (QX₁PX₃, orQX₁X₂[F,Y,W,I,L,V], where X₁ and X₃ are not proline) then a 16 mer wasdesigned, whereby the 12 mer with glutamine at position 7 was flanked bythe native residues at positions −1 and 13 and by glycine at positions−2 and 14. This strategy allowed the central, potentially deamidatedglutamine residue to be accommodated at anchor positions 4, 6 or 7 inany potential 9 mer HLA-DQ2-peptide-binding sequence. If selected 20mers did not include any 12 mer sequences with glutamine at position 7,then two 16 mers overlapping by 12 residues were synthesised. Somesecond round 16 mers with a central glutamine residue susceptible totTG-mediated deamidation were also synthesised with glutamine replacedby glutamate (in silico deamidation).

The wheat second round library consisted of 551 16 mers (including 113glutamate-substituted 16 mers) that were tested using PBMC from 34celiac disease donors after wheat challenge (including 26 responders),the barley library had eighty-nine 16 mers and included 9 substitutedwith glutamate that were tested using PBMC from 10 celiac disease donorsafter barley challenge (including 8 responders), and the rye library hadsixty-four 16 mers and included 11 substituted with glutamate that wastested using PBMC from 11 celiac disease donors after rye challenge(including 11 responders).

Hierarchy of stimulatory peptides was clearly demonstrated for eachgrain (see FIG. 6). Amongst the combined 652 20 mers in the gliadinPilot and 2723 20 mers in the Comprehensive libraries, 34 (1%) had ascore≧30, 300 (9%) had a score≧5, while 2111 had a score of 0. Onehundred and seventy-one of the 300 (57%) tTG-treated first round 20 merswith scores of ≧5 generated second-round tTG-treated 16 mers withscores≧5, and amongst these second round 16 mers there were 89 uniquesequences (see FIG. 7). These 89 confirmed T-cell stimulatory sequencesin the second round included 32 derived from gliadins, 1 from LMWglutenins, 4 from HMW glutenins, 30 from hordeins, and 29 from secalins,5 were common to prolamin families in two different grains and 1 was inthree prolamin families in all three grains.

All 89 confirmed T cell stimulatory 16 mers contained proline and/orglutamine.

Bioactivity following deamidation of second round peptides by tTG wasthe same as synthesising peptides with glutamate replacing glutamineresidues predicted to be susceptible to tTG (data not shown).

Exceptions to the requirement for deamidation were the closely relatedbut infrequently recognised HMW glutenin 16 mers W21 QGQQGYYPISPQQSGQ(SEQ ID NO:91), W22 QGQPGYYPTSPQQIGQ (SEQ ID NO:92), W24PGQGQSGYYPTSPQQS (SEQ ID NO:95), and W29 GQGQSGYYPTSPQQSG (SEQ IDNO:104), and the gliadin W36 QYEVIRSLVLRTLPNM (SEQ ID NO:116). Peptideswere considered “dominant” for a particular celiac donor if theyelicited at least 70% of the response of the most active peptide in eachlibrary for that donor. In the wheat, rye and barley second round, ten16 mers and thirty-one 12 mers with corresponding glutamate substitutedsequences (SEQ ID NOs:47, 48, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 95, 102, 103,104, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 136, 169, 170, 171, 172, 173, 174, 177, 178,179, 180, 183, 184, 187, 188, 189, 190, 191, 192, 209, 210) weredominant in at least 1 donor, while only four 16 mers and twenty-one 12mers (with corresponding glutamate-substituted variants) were dominantin more than 10% of donors (SEQ ID NOs:47, 48, 56, 57, 58, 59, 60, 61,62, 63, 64, 80, 81, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 136, 169, 170, 171, 172, 173, 174, 179, 180,183, 184, 187, 188, 191, 192). The highest scoring second round wheatgluten-, hordein- and secalin-derived 16 mers were dominant in more than50% and overall were recognised by more than 80% of donors.

The hierarchy and dominance of stimulatory peptides was strikinglydifferent according to the grain consumed (see FIG. 8). The stimulatorycapacity of peptides sharing the sequence motifQQPFPQPEQP(F,I)P(W,L,Y,Q)(Q,S) was not specific to any grain, ω-gliadin17 mer W03-E7 QPEQPFPQPEQPFPWQP (SEQ ID NO:55) was consistently the mostactive of this family and is the universal dominant T cell stimulatorypeptide in gluten. Other peptides were dominant almost exclusively afteronly one grain. For example, the α-gliadin 17 mer QLQPFPQPELPYPQPQP (SEQID NO:225; encompassing SEQ ID NO:62 (W02-E7) including DQ2-α-I (SEQ IDNO:3), and DQ2-α-II (SEQ ID NO:4)) was dominant only after wheat glutenchallenge, the hordein 16 mer B08-E7 PQQPIPEQPQPYPQQP (SEQ ID NO:318;encompassing SEQ ID NO:127 (B06-E7)) only after barley gluten challenge,and the secalin sequence QPFPQQPEQIIPQQ (SEQ ID NO:323; encompassing SEQID NO:190 (R11-E7)) only after rye gluten challenge. Other peptidesincluding the motif QPFP(W,L,Y,V,I)QPEQPFPQ elicited relatively strongerresponses after barley or rye than wheat gluten challenge. The “grainspecificity” of dominant T-cell stimulatory peptides provided afunctional definition for redundancy of T cell recognition thatcompliments the traditional approach to determination ofcross-reactivity based on T cell clones.

T cell clones were raised from intestinal biopsies or PBMC from celiacdisease donors to dominant deamidated peptides. The cytokine profiles ofT-cell clones were Th1 or Th0, and all were HLA-DQ2 restricted. Minimalcore sequences were determined using lysine scans of the parent peptide.T cell clones raised against NPL001 (SEQ ID NO:228) were specific forDQ2-α-I (SEQ ID NO:3) or DQ2-α-II (SEQ ID NO:4), and against NPL002 (SEQID NO:229) were specific for DQ2-ω-I PFPQPEQPF (SEQ ID NO:10) orDQ2-ω-II PQPEQPFPW (SEQ ID NO:15). Single T cell clones raised againstNPL003 (SEQ ID NO:230) were specific for DQ2-Hor-I PIPEQPQPY (SEQ IDNO:17), and the fully deamidated variant of SEQ ID NO:189,pyroEQPFPEQPEQIIPQQP-amide (SEQ ID NO:226; NPL004) (core 9 mer notdetermined, DQ2-SEC-I). One further clone raised against deamidatedgliadin was specific for W11-E7 QAFPQPEQTFPH (SEQ ID NO:74) (9 mer corenot determined). Each of the clones were screened against the secondround tTG-treated gliadin/glutenin, hordein, and secalin libraries andalso a further Verification 18 mer library (see Table 3) encompassingall unique 10 mers encoded by T. aestivum gliadins, H. vulgare hordeinsand S. cereale secalins in their wild-type sequence and with in silicodeamidation (glutamate replacing glutamine according to the tTGdeamidation motif). There was little cross-reactivity of clones fordominant stimulatory peptides, but substantial redundancy of peptiderecognition for many of sub-dominant gluten peptides. Altogether, 11clones specific for 6 epitopes, DQ2-α-I (SEQ ID NO:3), DQ2-α-II (SEQ IDNO:4), DQ2-ω-I (SEQ ID NO:10), DQ2-ω-II (SEQ ID NO:15), DQ2-Hor-I (SEQID NO:17), and DQ2-Sec-I (SEQ ID NO:226) present in 4 dominant T cellstimulatory peptides, W02-E7, W03-E7, B08-E2E7, and R11-E4E7 (SEQ IDNOs:62, 55, 319, 322 respectively) recognised 22/37 gliadin/glutenin,26/30 hordein, and 22/29 secalin sequences confirmed as stimulatorypeptides in FIG. 7.

IFNγ ELISpot assay using PBMC collected from HLA-DQ2⁺ celiac diseasedonors after gluten challenge with muffins made from an equal mixture ofwheat, barley and rye flour was used to compare the relative frequencyof T cells specific for W02-E7, W03-E7, B08-E2E7, and R11-E4E7 (SEQ IDNOs:62, 55, 319, 322 respectively), together with an atypical raredominant gliadin peptide W36 (SEQ ID NO:116) and an oat avenin homologueof Av-α9A QYQPYPEQEQPILQQ (SEQ ID NO:323; see FIG. 9A). The response tothe equimolar mixture of W02-E7, W03-E7, B08-E2E7 (SEQ ID NOs:62, 55,319; Cocktail 2) at an optimal concentration was no different from themixture of 6 peptides, but clearly greater than W02-E7 (SEQ ID NOs:62)and/or W03-E7 (SEQ ID NOs:55). When Cocktail 2 (50 μM) was assessedafter either wheat, barley or rye gluten challenge it stimulated IFNγELISpot responses equivalent to at least two-thirds of that stimulatedby optimal concentrations of tTG-treated gliadin, hordein, or ω-secalin(320 μg/ml), respectively (see FIGS. 9B, C, and D).

To improve their chemical stability and increase resistance toexopeptidases, peptides were synthesised as acetate salts of “capped”N-pyroglutamate, C-amide: NPL001 (SEQ ID NO:228), NPL002 (SEQ ID NO:229)and NPL003 (SEQ ID NO:230) 15 mers or 16 mers with glutamate at sitespredicted to be deamidated by tTG. Indeed, capping extended thehalf-lives of peptides from 10-12 minutes for free peptides: NPL033,NPL038, and NPL034 (SEQ ID NOs:13, 320 and 321) after bolus intradermalinjection of 0.9 mg in 0.1 ml in an adult rat to 26-28 minutes withN-pyroglutamate and C-amidation (SEQ ID NOs:228, 229 and 230) or 19-24minutes with N-acetylation and C-amidation (SEQ ID NOs:231, 232, and233), (see Table 4). Bioavailability, as measured by area under thecurve analysis, was also substantially increased by as much asthirty-four times with addition of N-pyroglutamate or N-acetyl, andC-amidation capping.

TABLE 4 Pharmacokinetics of derivatised T-cell stimulatory peptides.Free N- and C-terminals N-Acetyl and C-amide N-pyroGlu and C-amideLQPFPQPELPYPQPQ N-Acetyl- pyroE- (SEQ ID NO: 13) QLQPFPQPELPYPQPQ-LQPFPQPELPYPQPQ- NPL033 amide amide T1/2 10.2 minutes (SEQ ID NO: 231)(SEQ ID NO: 228) AUC 2618 NPL030 NPL001 T1/2 19.4 minutesT1/2 28.20 minutes AUC 43474 AUC 89350 PQQPFPQPEQPFPWQP N-Acetyl- pyroE-(SEQ ID NO: 320) QQPFPQPEQPFPWQP- QPFPQPEQPFPWQP- T1/2 13.2 minutesamide amide AUC 22393 (SEQ ID NO: 232) (SEQ ID NO: 229) NPL031 NPL002T1/2 22.9 minutes T1/2 27.18 minutes AUC 80263 AUC 81514 N-Acetyl-pyroE- FPEQPIPEQPQPYPQQ FPEQPIPEQPQPYPQQ- PEQPIPEQPQPYPQQ-(SEQ ID NO: 321) amide amide T1/2 12.5 minutes (SEQ ID NO: 233)(SEQ ID NO: 230) AUC 8206 NPL032 NPL003 T1/2 24.2 minutesT1/2 25.98 minutes AUC79439 AUC 51390 T1/2 half life, and AUC area underthe curve (bioavailability) after intradermal bolus injection 0.9 mg in0.1 ml saline of equimolar mixture of NPL001 + 2 + 3, NPL033 + 38 + 34,or NPL030 + 31 + 32

The inventors findings support the notion that peptides encompassingepitopes present in NPL001 (SEQ ID NO:228), NPL002 (SEQ ID NO:229) andNPL003 (SEQ ID NO:230), are dominant, non-redundant, and consistentlycontribute a substantial proportion of the T-cell stimulatory activityof gluten. These 3 peptides or the epitopes within them are thereforelikely to be critical to the design of a peptide-based therapeuticvaccine or in functional diagnostics that are consistently applicable toHLA-DQ2-associated celiac disease.

These findings emphasise that in vitro approaches reliant on expansionof rare antigen-specific T cells frequently do not necessarily translateto epitopes relevant in vivo after acute disease reactivation. Indeedthe majority of non-redundant dominant T cell stimulatory peptidesidentified in the present study have not been previously described infunctional studies utilising T cell clones and lines. Sincecomprehensive epitope mapping using T cells elicited in vivo by thepathogenic antigen has not been carried out previously, this studyprovides the first true test of an in vitro approach to mapping epitopesrelevant to an immune human disease. The prior art does not describe themanner in which non-redundant dominant T cell stimulatory peptides wouldbe selected for peptide-based immunotherapy to maximise the number of Tcells targeted in the greatest number of patients while also minimisingthe number of peptides to simplify formulation.

However, additional peptides are likely to add to the T cell stimulatorycapacity and consistency of donor T cell responses of this mixture afterwheat, barley or rye challenge. Gluten peptides with the highest“scores” but not recognised by T cell clones specific for DQ2-α-I (SEQID NO:3), DQ2-α-II (SEQ ID NO:4), DQ2-ω-I (SEQ ID NO:10), DQ2-ω-II (SEQID NO:15), or DQ2-Hor-I (SEQ ID NO:17) are the most likely to addfurther to the T cell stimulatory capacity of the mixture. Increasingthe proportion of gluten-specific T cells consistently targeted by apeptide mixture is likely to improve its therapeutic or diagnosticutility for HLA-DQ2⁺8⁻ celiac disease, but may also complicateformulation, compromise chemical stability, and increase the likelihoodof adverse effects.

On the other hand, NPL001 (SEQ ID NO:228) could be substituted with asingle peptide, for example, including the sequence LPYPQPELPYPQ (SEQ IDNO:60; W01-E7) recognised by T cell clones specific for DQ2-α-I (SEQ IDNO:3), and also T cell clones DQ2-α-II (SEQ ID NO:4). Alternatively,NPL001 (SEQ ID NO:228) could be substituted for two separate peptides,one recognised by T cell clones specific for DQ2-α-I (SEQ ID NO:3), andthe other recognised by T cell clones specific for DQ2-α-II (SEQ IDNO:4). The same principle could be applied to NPL002 (SEQ ID NO:229) andNPL003 (SEQ ID NO:230). This may be advantageous to improve formulationand stability.

Example 3: NexVax2 in Mouse Model

The optimal administration and dose regimen of a peptide-basedtherapeutic vaccine to induce clinical tolerance to gluten and remissionof celiac disease while consuming gluten is not known. However, anessential property of any peptide-based therapeutic would be its abilityto activate cognate T cells in the target organ in vivo.

The interaction between NPL001 (SEQ ID NO:228) and cognate T cells invivo has been modelled by developing transgenic Black-6 mice expressingfunctional HLA-DR3 and -DQ2 (but not murine MHC Class II molecules) onantigen presenting cells (APC) who are transferred 3×10⁶ CFSE labelledCD4⁺ T cells specific for NPL001 (Chen Z., et al., 2006). The donormouse (HH8-1) is transgenic for the NPL001-specific T cell receptor andhuman CD4 expressed on T cells, and also expresses HLA-DR3 DQ2 on APC.Overall 96% of the CD4⁺ T cells in the HH8-1 mouse are clonal andspecific for NPL001 (results not shown).

Four days after subcutaneous administration (in the hind foot hock) ofan equimolar mixture of NPL001, NPL002 and NPL003 in 50 μl saline,spleen, gut-draining mesenteric lymph nodes (MLN) and the local drainingpopliteal lymph nodes (PLN) are harvested. Isolated mononuclear cellsare stained for hCD4, and the T-cell receptor α- and β-chains expressedon the HH8-1 NPL001-specific T cells (Vα8 and Vβ8). Proliferation oftransferred CFSE labelled cells is measured as the % of CFSE^(pos) cellshaving undergone one or more division, as indicated by dilution of CFSEstaining. FIG. 10 shows dose dependent proliferation of T cells specificfor NPL001 is observed following subcutaneous administration of between0.9 and 30 μg, half-maximal response is achieved with 5 μg. No clinicaltoxicity is observed with these or doses as high as 900 μg, despite theT cells having a Th1 phenotype and secreting IFNγ upon stimulation withNPL001.

This mouse model has the potential to allow the demonstration of (i)proof-of-principle, (ii) mechanism of action and (iii) optimisation ofdose regime for the induction of tolerance following administration ofNexVax2 therapeutic vaccine (an equimolar mixture of NPL001, NPL002 andNPL003 in saline). In the previous mouse studies the inventors havedemonstrated that a single dose of NexVax2, or the relevant peptidecomponent NPL001, is bioactive in vivo. Administration of NPL001 inducesproliferation of HH8-1 gliadin-specific T cells in an adoptive transfermodel at the highest dose to be administered in Phase 1b human clinicaltrials. The dose response for the activation of transgenicNPL001-specific T cells was subsequently determined. Based on thispreliminary data, the ability of NexVax2 therapeutic vaccine to modulategliadin-specific T cell responses and mechanism of action can beaddressed in a biologically relevant mouse model.

The objective of the study was to determine whether repeatadministration of the therapeutic vaccine, NexVax2, using a regimedesigned to induce immunological tolerance is capable of modulating thegliadin-specific T cell response in a gliadin-specific TCR-Tg mousemodel.

Animals were identified, allocated to experimental groups and treated asin Table 5 below.

TABLE 5 Allocation of animals to experimental groups. Mouse Number GroupDose of NexVax2 Number of Doses Number per group A 10 μg 14 daily dose5A1, 5A2 2 B 3 μg 14 daily dose 5B1, 5B2 2 C 1 μg 14 daily dose 5C1, 5C22 D 0.3 μg 14 daily dose 5D1, 5D2 2 E 0 (Saline control) 14 daily dose5E 1 F 10 μg 1 dose on final 5F 1 day of treat- ment regime

The intradermal/subcutaneous route of administration was selected asthis is the intended route of administration in man. The dosage wasselected to cover the dose-response range that resulted in stimulationof all glaidin-specific T cells in the adoptive transfer model (10 μg)to low dose (0.3 μg) that did not result in proliferation ofCFSE-labelled gliadin-specific TCR-Tg T cells in the previous study(Nexpep3).

All peptides were GMP grade. The formulations were prepared by NexpepPty Ltd, and the concentration of peptide was adjusted for purity.NexVax2 consists of 3 peptides (NPL001, NPL002 and NPL003) each at 6mg/ml in saline.

The stated dose is the quantity of each peptide in NexVax2, not thetotal peptide concentration (i.e., 10 μg NexVax2 contains 10 μg NPL001,10 μg NPL002 and 10 μg NPL003). NPL001 was provided at 6 mg/ml insaline. Peptides were stored at −80° C. prior to injection.

Animals and Management

All experiments were carried out with the approval of the University ofMelbourne Animal Ethics Committee, AEC Register No 0707287.

Fourteen female HH8-1 and 4 hCD4.IAE^(−/−).DR3.DQ2 transgenic mice onthe C57BL/6 background were used. All mice were bred at the Universityof Melbourne, Department of Microbiology and Immunology Animal Facility.Mice were raised on a gluten-free diet (SF07-036) supplied by SpecialtyFeeds Pty Ltd, Perth Western Australia. Each animal was numbered byear-punching according to the Animal Facility protocol which identifiedit individually within the study and which corresponded to that animal'snumber. The animals were housed individually or in groups of up to 4mice in cages with stainless steel grid tops and solid bottoms. Woodshavings were used as bedding, and tissues supplied for nestingmaterial. Each cage was supplied with a water bottle containingacidified water and food hopper containing gluten-free mouse food. Theroom was maintained between 21° C. and 24° C. The range for relativehumidity was 37-58%. A 12 hour light/dark cycle was in operation (lighthours 0700-1900) with a minimum of 15 air changes per hour.

NexVax2 was diluted to 200 μg/ml in sterile saline, aliquoted and storedat −80° C. for use. For each treatment, an aliquot was thawed anddiluted in sterile saline. Groups of female HH8-1 mice (n=2) wereinjected subcutaneously on the flank with 50 μl containing a titratingdose of NexVax2 (10 μg, 3 μg, 1 μg and 0.3 μg) diluted in saline orsaline alone. Mice were injected daily for 14 days. One mouse received asingle dose of 10 μg NexVax2 on the final day of the treatment regime.

Mice were monitored daily for swelling or irritation at the injectionsite, symptoms of adverse systemic response (hunched or ruffledappearance, lethargy, shivering, moribund). The onset, intensity andduration of any signs were recorded.

A blood sample was collected from the retro-orbital sinus prior toadministration of peptide and by cardiac puncture following CO₂euthanasia at the completion of the experiment. Blood was stored at 4°C. overnight, the clot removed and serum collected followingcentrifugation. Sera were stored at −80° C. for future analysis ifrequired.

Mice were killed by CO₂ euthanasia 3 days after the final administrationof peptide and the spleens were collected. Single cell suspensions wereprepared by sieving through 70 μm nylon mesh cell strainers. Red bloodcells were removed from spleens by Tris ammonium chloride lysis. CD4⁺ Tcells were isolated by negative depletion using the CD4⁺ T cellisolation kit (Miltenyi Biotech) according to the manufacturer'sinstructions. Gliadin-specific T cells were enriched from the spleens of4 naïve HH8-1 mice using the same protocol. APC were prepared from thespleens of three hCD4.IAE^(−/−).DR3-DQ2 transgenic mice. Single cellsuspensions were prepared as above. Splenocytes were gamma irradiated(2,200 rads) before use as APC.

Cells were phenotyped by antibody staining and FACS analysis.Gliadin-specific CD4⁺ T cells were identified by staining with TCR Vα8.3and human CD4, and surface stained with anti-CD25 and anti-GITRmonoclonal antibodies. Intracellular FoxP3 expression was determinedusing a FoxP3 staining kit (eBiosciences) according to themanufacturer's instructions. Samples were fixed in FACS fixative (1%paraformaldehyde, 2% glucose in PBS) and analysed by flow cytometry onthe LSR II (BD Bioscience). IFNγ and IL-10 producing T cells wereidentified by intracellular cytokine staining following stimulation withPMA/Ionomycin.

Briefly, 1×10⁶ splenoctyes from treated mice were cultured for 6 hoursin complete DMEM (DMEM supplemented with 10% heat inactivated foetalcalf serum, 2 mM glutamine, non-essential amino acids, 50 μM2-mercaptoethanol, penicillin and streptomycin) and 5 μg/ml Brefeldin Awith or without 50 ng/ml PMA and 500 ng/ml Ionomycin. Cells were thenstained for the surface molecules (TCR Vβ8.3 and human CD4), washed thenfixed with 1% paraformaldehyde/30 minutes, washed twice and thenincubated with anti-IFNγ or anti-IL-10 antibody diluted in PBScontaining 0.2% Saponin. Samples were analysed by flow cytometry on theLSR II (BD Bioscience) gating on TCR Vβ8.3⁺, human CD4⁺ lymphocytes.

2×10⁴ purified T cells from each mouse were cultured in triplicate inround-bottomed 96 well plates in complete DMEM with 3×10⁵gamma-irradiated APC in the presence or absence of 2 μg/ml NPL001 at 37°C./5% CO₂. Following 72 hours of culture, supernatants were collectedand stored at −80° C. for analysis of the cytokine secretion.

Samples were tested for the presence of mouse IL-2, IL-4, IL-5, IL-6,IL-10, IL-12p70, TNFα and IFNγ by cytometric bead array flexset (CBA, BDBioscience) according to the manufacturers' instructions. Samples wereanalysed by flow cytometry on the FACS Canto (BD Biosciences), and dataanalysed using FCAP Array software (BD Bioscience).

Supernatants from cultured splenocytes were tested neat and at 1:10dilution. The concentration of cytokine was determined against theprovided standards diluted from 2500-10 pg/ml.

2×10⁴ purified CD4 T cells from NexVax2-treated HH8-1 mice were culturedwith irradiated syngeneic spleen cells (2,200 rads, 3×10⁵/well) intriplicate assays in the presence of 0, 0.02, 0.2, 2 or 10 μg/ml NPL001peptide. For suppression assays, 2×10⁴ naïve HH8-1 CD4 T cells(responders) were cultured with an equal number (1:1) of CD4⁺ T cellsfrom NexVax2-treated mice, titrated NPL001 peptide and irradiatedsyngeneic spleen cells (2,200 rads, 3×10⁵/well) in triplicate assays. Ina separate assay naïve responders were cultured with CD4⁺ T cells fromNexVax2-treated mice at responder:suppresssor ratios of 1:1, 3:1 and 9:1in the presence of sub-optimal concentration of NPL001 peptide (0.2μg/ml) and APC.

T cell proliferation was measured by the addition of 1 μCi ³H-thymidinefor the last 24 hours of the 96 hour cultures. Results are recorded ascounts per minute (cpm), with the mean of each triplicate plotted anderror bars representing the standard deviation.

RNA was extracted from 5×10⁵-2×10⁶ purified T cells from NexVax2 treatedmice using RNAeasy Plus™ RNA extraction kit (QIAGEN) according to themanufacturers instructions. RNA was stored at −80° C. for futureanalysis if required.

Mice were monitored daily for any apparent adverse response followingtreatment. There were no unscheduled deaths during the observationperiod. There were no systemic adverse signs noted in any animal duringthe observation period. All mice remained apparently healthy, with noobservable decline in activity or appearance. No local inflammation atthe site of injection was observed in the mice immunised with peptide insaline or saline alone.

Phenotype Analysis

Peptide immunotherapy has been associated with the induction ofperipheral tolerance mediated by the induction of thymically derived orde novo generated CD4^(+CD)25⁺ FoxP3⁺ regulatory T (Treg) cells.Additionally, such induction is associated with generation of IL-10secreting peptide induced Treg cells. The effect of repeatadministration of NexVax2 on the number and phenotype of splenicgliadin-specific T cells was determined. Gliadin-specific T cells in thespleen were identified by TCR Vβ8.3 and CD4 expression. The proportionof gliadin-specific T cells in the lymphocyte gate and the total numberper spleen was determined. See FIG. 11, which shows repeatadministration of NexVax2 leads to the reduction in the proportion (A)and number (B) of gliadin-specific CD4⁺ T cells in the spleen. HH8-1gliadin-specific TCR transgenic mice were injected subcutaneously dailyfor 14 days with the indicated amount of NexVax2. Spleens were harvested3 days after the final injection, processed and stained with antibodiesto identify transgenic T cells (Vβ8.3 and hCD4). The total number oftransgenic T cells was calculated from the total cell spleen cellcounts. Dots indicate individual mice.

Treatment with multiple doses NexVax2 at the highest dose tested (10 μg)resulted in an apparent decrease in both the proportion and number ofgliadin-specific T cells by approximately 50-65%, suggesting eitherantigen-induced cell death or recruitment of these cells away from thespleen.

In order to determine whether the repeat administration of NexVax2induced a Treg population, gliadin-specific T cells were identified byTCR Vβ8.3 and CD4 expression and the proportion of these expressing CD25and FoxP3 (see FIG. 12A) or CD25 and GITR (see FIG. 12B) determined.FIG. 12 shows repeat administration of NexVax2 leads to the induction ofTreg cells. HH8-1 gliadin-specific TCR transgenic mice were injectedsubcutaneously daily for 14 days with the indicated amount of NexVax2.Spleens were harvested 3 days after the final injection, processed andstained with antibodies to TCR Vα8.3, CD4, CD25, FoxP3 and GITR. FACSplots of gliadin-specific, CD4 lymphocytes expression CD25 and FoxP3 (A)or CD25 and GITR (B) are shown. Treatment with multiple doses of 10 μgor 3 μg NexVax2 resulted in an increased proportion of gliadin-specificTreg cells in the spleen in a dose-dependent manner.Glucocorticoid-Induced TNF Receptor (GITR) is expressed predominantly onCD25⁺ Treg cells. Staining revealed that the CD25⁺ population ofgliadin-specific T cells co-expressed GITR. The percentage of GITR cellsincreased in proportion with the expression of CD25 following NexVax2administration.

The proportion of gliadin-specific T cells with the capacity to produceIFNγ or IL-10 directly ex-vivo in response to non-specific activationwas examined. Splenocytes were cultured with and without PMA/Ionomycinin the presence of Brefeldin A. IFNγ and IL-10 production bygliadin-specific T cells was determined by flow cytometry. FIG. 13 showsrepeat administration of NexVax2 results in an increase in theproportion of IFNγ and IL-10 producing cells directly ex vivo. HH8-1mice received daily subcutaneous. administration of 10, 3, 1, or 0.3 μgNexVax2 in saline or saline alone for 14 days, or a singleadministration of 10 μg NexVax2 on day-14. Three days after the finalinjection, mice were killed and the proportion of splenic TCRVβ8.3/hCD4⁺ cells expressing IFNγ (A) or IL-10 (B) was determined byintracellular cytokine staining and flow cytometry following a 6 hourincubation in the presence or absence of PMA/Ionomycin. Dots representindividual mice and the dotted line indicates the proportion of cytokinepositive cells in naïve HH8-1 mice.

Repeat administration of 10 μg NexVax2 resulted in an increasedproportion of IFNγ producing gliadin-specific T cells, and a small butconsistent increase in the proportion of IL-10 producinggliadin-specific T cells. Repeat administration of 1 or 3 μg NexVax2resulted in an increase in the frequency of IFNγ producing T cells inone of the two mice tested in each group.

Proliferative Response to Peptide

The proliferative capacity of gliadin-specific T cells following repeatadministration of NexVax2 was examined in order to determine whetherthese cells have an anergic phenotype. Failure to proliferate in vitrois a feature of both CD25⁺/FoxP3⁺ Treg cells and IL-10 producingpeptide-induced Treg cells. This reduction in the ability to proliferateis reversible by the addition of IL-2 to cultures.

Purified CD4⁺ splenic T cells were cultured in the presence ofgamma-irradiated APC from hCD4.IAE^(−/−).DR3.DQ2 transgenic mice andgraded concentrations of cognate peptide, NPL001. Proliferation wasmeasured by the incorporation of ³H-Thymidine for the final 24 hours ofthe 4 day culture (see FIG. 14A). FIG. 14 shows the proliferativecapacity of gliadin-specific T cells to cognate antigen is diminishedfollowing repeat administration of NexVax2 and restored in the presenceof IL-2. HH8-1 mice received daily subcutaneous administration of 10, 3,1, or 0.3 μg NexVax2 in saline or saline alone for 14 days, or a singleadministration of 10 μg NexVax2 on day-14. Three days after the finalinjection, mice were killed and CD4⁺ T cells purified and cultured withNPL001 peptide and irradiated APCs in the presence or absence of 10 U/mlIL-2. After 72 hours, wells were pulsed with 1 μCi 3H-Thymidine for 24hours and plates were harvested and counted.

A. Proliferative response of mice treated with indicated dose of NexVax2to 0.2 μg/ml NPL001 peptide.

B. Proliferative dose-response of naïve and repeat administration of 10μg NexVax2 to NPL001.

Error bars represent standard deviation of triplicate cultures.

T cells from antigen naïve, saline-treated mice proliferated well inresponse to NPL001, whereas T cells from NexVax2-treated mice showed asubstantial reduction in their capacity to respond to NPL001,particularly evident at sub-optimal peptide concentration (0.2 μg/ml).Repeat administration with 10 μg NexVax2 led to a 90-97% reduction inthe proliferative response to 0.2 μg/ml NPL001. The reduction inproliferation was dose dependent, and even the lowest dose administered(0.3 μg) resulted in a 20-37% reduction in proliferation at sub-optimalpeptide concentrations. The addition of 10 U/ml IL-2 to the culturesinduced a low level of proliferation in the absence of peptide(approximately twice the background), however in the presence of peptidethe unresponsive state of T cells from NexVax2-treated mice wasreversed, such that the response of peptide-treated mice was theequivalent to the saline-treated control.

Failure to proliferate in response to NexVax2 administration wasobserved over a range of doses (see FIG. 14B). This was particularlyevident following administration of the highest dose of NexVax2 (10 μg)and was less effective following treatment with lower NexVax2 doses,particularly in response to maximal peptide stimulation (data notshown).

Suppression of Naïve HH8-1 T Cell Activation

The observed failure to proliferate could be a result of an anergicphenotype, where the T cells themselves have become less sensitive toantigen stimulation, or due to the presence of a Treg population. Thus,the ability of treatment with NexVax2 to generate a Treg populationcapable of suppressing the proliferative response of naïvegliadin-specific T cells to NPL001 peptide in in vitro culture wasassessed.

FIG. 15 shows T cells from mice treated with NexVax2 are able tosuppress the proliferation of naïve gliadin-specific T cells. HH8-1 micereceived daily subcutaneous. administration of 10, 3, 1, or 0.3 μgNexVax2 in saline or saline alone for 14 days, or a singleadministration of 10 μg NexVax2 on day-14. Three days after the finalinjection, purified CD4⁺ T cells from treated mice (suppressors) wereco-cultured with T cells untreated HH8-1 mice (responders), NPL001peptide and irradiated APC. After 72 hours, wells were pulsed with 1 μCi3H-Thymidine for 24 hours and plates were harvested and counted. In FIG.15A, T cells from 10 μg NexVax2×14-treated mice (left panel) orsaline-treated mice (right panel) were co-cultured with an equal numberof naive HH8-1 CD4⁺ T cells and titrated NPL001 peptide. In FIG. 15B, aconstant number of naïve HH8-1 T cells (2×104) were co-cultured withtitrated numbers of NexVax2-treated T cells (2×10000, 6.6×1000,2.2×1000) and 0.2 μg/ml NPL001. The average inhibition of naïveresponder proliferation was calculated from the 2 mice in each treatmentgroup. Error bars represent standard deviation of triplicate cultures.

Purified T cells from NexVax2 treated mice were co-cultured with naïveHH8-1 gliadin-specific T cells at a 1:1 ratio in the presence of atitrated dose of NPL001 (see FIG. 15A) or at responder:suppresssor ratioof 1:1, 3:1 or 9:1 in the presence of 0.2 μg/ml NPL001 (see FIG. 15B).Suppression of responder cell proliferation was observed followingtreatment with the repeat administration of 10 μg or 3 μg NexVax2 and atresponder:suppresssor ratio of 1:1 over a range of stimulatory peptideconcentrations. This result indicates the presence of a regulatorypopulation. Given that phenotyping demonstrated an increase in theproportion of gliadin-specific Treg cells only in mice treated with the10 or 3 μg of NexVax2 and that Treg cells comprised between 7 and 18% ofthe total gliadin-specific population, the inhibition of naïve HH8-1 Tcell proliferation observed is within expectations.

Cytokine Profile Following In Vitro Culture

Immune modulation can alter the cytokine profile of responder cells. Forexample, intranasal administration of peptide has been shown to generateIL-10 secreting peptide-induced Treg cells. The profile of cytokineproduction by CD4⁺ T cells from gliadin-specific TCR transgenic micethat had been treated with repeat administration of graded amounts ofNexVax2 was examined following in vitro culture in the presence orabsence of 2 μg/ml NPL001 and irradiated syngeneic APC. Supernatantsfrom day-3 of culture were collected and assessed for the production ofTh1-associated cytokines (IL-2, IFNγ, IL-12 and TNFα) and Th2-associatedcytokines (IL-4, IL-5, IL-6 and IL-10) (see FIG. 16). FIG. 16 shows invitro cytokine production. CD4⁺ T cells were purified from the spleen ofHH8-1 mice that received daily subcutaneous administration of 10, 3, 1,or 0.3 μg NexVax2 in saline or saline alone for 14 days, or a singleadministration of 10 μg NexVax2 on day-14. 3×104 CD4 T cells werecultured in the presence of 2 μg of NPL001 (▪) or without peptide (□)and 3×100000 gamma-irradiated APC. Supernatant were harvested at 72hours and tested by cytometric bead array for the production of Th1cytokines (IL-2, IFNγ, IL-12, TNFα), and Th2 cytokines (IL-4, IL-5 IL-6and IL-10). Results show the average cytokine production of the two micein the treatment groups. No IL-12, IL-4 or IL-5 was detected in thesupernatants of the cultures. A marked reduction in the production ofIL-2, IFNγ, and TNFα was observed from the cultures of mice receivingrepeat administration of 10 μg NexVax2. This reduction in cytokineproduction closely reflects the reduced proliferative response to NPL001peptide in culture. In addition the T cells from mice receiving repeatinjections of 10 μg NexVax2 produced a 3.5 fold increased amount ofIL-10 following peptide stimulation in vitro, suggesting potentialskewing towards an IL-10 producing Treg phenotype in these mice.

This experiment was designed to determine whether repeat administrationof the therapeutic vaccine NexVax2, using a regime designed to induceimmunological tolerance, is capable of modulating the gliadin-specific Tcell response in a gliadin-specific T cell receptor-transgenic mousemodel. NexVax2 was administered via subcutaneous injection of peptide insaline over 14 consecutive days. This treatment resulted firstly in anapparent reduction in the number of gliadin-specific T cells in thespleen. The remaining T cells showed a reduction in their proliferativeresponse to their cognate antigen, which was reversed in the presence ofIL-2 suggesting an “anergic” phenotype or the presence of a Tregpopulation. This reduced proliferative response was accompanied by areduction in the amount of Th1 cytokines produced in culture, and by anincrease in IL-10 production. An increase in gliadin-specific IL-10producing cells was also observed directly ex-vivo along with anincrease in the total number and the proportion of FoxP3⁺, GITR⁺ Tregcells. In co-culture experiments, T cells from treated mice were capableof suppressing the proliferative response of naïve gliadin-specific Tcells responding to NPL001 peptide.

The repeat administration of NexVax2 at the highest dose tested (10 μgper day, over 14 consecutive days) demonstrated the modulation of theresponse of gliadin-specific T cells from treated gliadin-specific Tcell receptor transgenic mice.

The results provide evidence that the subcutaneous administration ofNexVax2 peptide in saline is capable of modifying the T cell response tothe immunodominant gliadin peptide using a biologically relevantTCR-transgenic mouse model.

Example 4: NexVax2 Vaccine for Human Celiac Disease

The NexVax2 vaccine was prepared in GMP form for administration to humanpatients with celiac disease.

Phase I Study to Determine Safety, Tolerability and Bioactivity ofNexVax2 in HLA-DQ2⁺ Volunteers with Celiac Disease Following aLong-Term, Strict Gluten-Free Diet.

Objectives

The primary objective of this study was:

-   -   To evaluate the safety and tolerability of weekly injections of        NexVax2 administered intradermally for 3 weeks.

The secondary objectives of this study were:

-   -   To determine the bioactivity of NexVax2 following 3 weekly doses        in celiac disease volunteers through the measurement of T-cell        response as assessed by T cell frequency and cytokine release.    -   To determine the bioactivity of NexVax2 following 3 weekly doses        in celiac disease volunteers through the measure of symptomatic        response after gluten challenge.    -   To measure the pharmacokinetics of NexVax2 following a single        intradermal injection in celiac disease volunteers.    -   To measure the induction of antibodies specific for NexVax2        following 3 weekly doses in celiac disease volunteers.

Study Design

A Phase I, single-centre, placebo-controlled, dose-escalating study ofthe safety, tolerability and bioactivity of NexVax2 in celiac diseasevolunteers when administered weekly via intradermal injection.

Celiac disease patients were required to attend nine outpatient visits.This included three 8 hour visits to receive intradermal injections ofNexVax2 (over 3 weeks) and three 6 hour visits to undergo a standardgluten challenge.

Volunteers remained on study for approximately 25 days from the date ofthe first injection.

Study Population

Individuals with a diagnosis of celiac disease according to acceptedEuropean Society of Paediatric Gastroenterology, Hepatology andNutrition diagnostic criteria (Walker-Smith et al., 1990) following astrict gluten free diet, who possess genes encoding HLA-DQ2 (DQA1*05 andDQB1*02) but not HLA-DQ8 (DQA1*03 and DQB1*0302).

Test Formulation for NexVax2

NexVax2 for injection, contained an equimolar (0.159 μmole per 100 μl,approx. 3 mg/ml) mixture of each of NPL001, NPL002 and NPL003 in a 0.9%normal saline sterile solution supplied by Nexpep Pty Ltd

Placebo Formulation

Sterile normal saline 0.9% supplied by Nexpep Pty Ltd.

Study Treatments

Cohort 1: comprising 2 sentinels, 1 dosed with 9 μg NexVax2 byintradermal injection and 1 dosed with placebo and a further 6 subjects,5 dosed with 9 μg NexVax2 and 1 dosed with placebo on days 1, 8 and 15.Cohort 2: as per Cohort 1 but subjects dosed with 30 μg NexVax2Cohort 3: as per Cohort 1 but subjects dosed with 90 μg NexVax2Cohort 4: as per Cohort 1 but subjects dosed with 60 μg NexVax2

Schedule for Dosing, Meals and Blood Collection

After fasting from midnight the evening prior to study drugadministration, the schedule for dose administration, meals,pharmacodynamic assessments, gluten challenge and blood collection(assuming a 0800 hour dosing time) was as shown in FIG. 17.

Assessments

-   -   Resting heart rate, semi-supine systolic/diastolic blood        pressure, respiratory rate and temperature was monitored: at        Screening; nominally at 0700 hours prior to receiving treatment        on days-1, 8 and 15 and at 4 hours post-dose; and on days-22, 23        and 24 prior to receiving the gluten challenge and on day-25 end        of study.    -   Blood samples for PBMC IFNγ ELISpot assay to enumerate the        frequency of NexVax2 specific T cells were collected on days-1,        6, 15, 20 and 25 (End of Study).    -   Blood samples for Bioplex analysis to determine PBMC cyotkine        release in response to NexVax2 were collected on days-1, 6, 15,        20 and 25 (End of Study).    -   PBMC were collected on days-1, 6, 15, 20 and 25 (End of Study)        and frozen for later assay of T cell function.    -   Serum was collected on days-1 and 20 for assessment of        antibodies specific for NexVax2.    -   Blood samples for pharmacokinetics sampling were collected on        day-15 at pre-dose and at 15, 30, 45, 60, 75, 90 minutes, 2        hours and 3 hours post-dose.    -   Clinical laboratory measures (biochemistry, urinalysis and        haematology) were performed: at Screening; on days-1, 8 and 15        pre-dose and 4 h post-dose; and pre-gluten challenge on days-20        and 22, and post gluten challenge and on day-25 (End of Study).    -   Pregnancy (urine) testing was conducted at Screening, pre-dose        on day-1, 8 and 15, and pre-gluten challenge on days-20, 21 and        22 and at end of study (day-25).    -   Urine drugs of abuse testing was conducted at Screening and        pre-dose on days-1, 8 and 15.    -   ECGs were performed: at Screening; nominally at 0700 hours prior        to receiving treatment on days-1, 8 and 15 and at 4 hours        post-dose; and on days-20, 21 and 22 prior to receiving the        gluten challenge and on day-25 end of study.

Data Analysis

Screening, Compliance and Safety Data

Demographics will be tabulated and summarised. Physical examination(including height and weight) at baseline and follow up andmedical/surgical history data at baseline will be listed. All clinicalsafety and tolerability data will be listed for each subject.

Laboratory values outside the laboratory's normal ranges will be listedseparately, with comments as to their clinical significance. Associatedrepeat values will be listed together. Vital sign measurements (restingheart rate, semi-supine systolic/diastolic blood pressure, respiratoryrate, temperature) and ECG parameters will be tabulated and summarised.

Tolerability Data

Treatment-emergent adverse events will be listed and summarised. Alladverse events reported in this study will be coded using MedDRA.

Immunological Assays

The inventors consider that a single treatment of NexVax2 will increasethe frequency of NexVax2-specific T cells in PBMC and will increasesecretion of cytokines and chemokines by mononuclear cells.

The inventors consider that PBMC drawn after repeated (3 weekly)injection of NexVax2 will have a lower frequency of NexVax2-specific Tcells than prior to treatment.

The inventors consider that compared to placebo-treated celiac diseasevolunteers, repeated (3 weekly) injection of NexVax2 will reduce thefrequency of T cells specific for NexVax2 and cytokine secretionstimulated by NexVax2 in PBMC collected 6 days after commencing 3-dayoral gluten challenge with wheat bread.

Ordinal data will be analysed by one-tailed paired Wilcoxon rank-sumtest. Normally distributed data will analysed by paired t-test. Ap-value<0.05 will be considered significant.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/118,643, the entirecontents of which are incorporation herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

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1. An agent comprising i) a first peptide comprising the amino acidsequence LQPFPQPELPYPQPQ (SEQ ID NO:13), or a biologically activefragment or variant thereof, ii) a second peptide comprising the aminoacid sequence QPFPQPEQPFPWQP (SEQ ID NO:14), or a biologically activefragment or variant thereof, and iii) a third peptide comprising theamino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO:16), or a biologicallyactive fragment or variant thereof.
 2. The agent of claim 1, wherein thesecond peptide comprises the amino acid sequence PQQPFPQPEQPFPWQP (SEQID NO:320), or a biologically active fragment or variant thereof, and/orthe third peptide comprises the amino acid sequence FPEQPIPEQPQPYPQQ(SEQ ID NO:321), or a biologically active fragment or variant thereof.3. The agent of claim 1, wherein the first, second and/or third peptidescomprise an N terminal acetyl group or pyroglutamate group, and/or a Cterminal amide group.
 4. The agent of claim 3, wherein the first, secondand/or third peptides comprise an N terminal pyroglutamate group and a Cterminal amide group.
 5. The agent of claim 1, wherein the first, secondand/or third peptides are conjugated to a compound.
 6. The agent ofclaim 5, wherein the compound is an adjuvant, or an MHC molecule orbinding fragment thereof.
 7. The agent of claim 1, wherein two or threeof the first, second and third peptides, or the biologically activefragment or variant of one or more thereof, are on a single polypeptidechain.
 8. The agent of claim 1 comprising one or more additionalpeptides comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:47, 48, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 95, 102, 103,104, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 136, 169, 170, 171, 172, 173, 174, 177, 178,179, 180, 183, 184, 187, 188, 189, 190, 191, 192, 209, 210, or abiologically active fragment or variant of any one or more thereof. 9.An agent comprising one or more polynucleotides encoding the agent ofclaim
 1. 10. An agent comprising i) a first peptide or a polynucleotidethat encodes the first peptide, ii) a second peptide or a polynucleotidethat encodes the first peptide, and iii) a third peptide or apolynucleotide that encodes the first peptide, wherein the first peptidecomprises the amino acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 13), or abiologically active fragment or variant thereof; the second peptidecomprises the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 14), or abiologically active fragment or variant thereof; and the third peptidecomprises the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 16), or abiologically active fragment or variant thereof.
 11. A substantiallypurified and/or recombinant peptide comprising an amino acid sequence asshown in any one or more of SEQ ID NO:16, 69, 73, 75, 78, 80, 87, 91,92, 95, 96, 98, 100, 104, 107, 113, 116, 117, 123, 138, 144, 147, 149,153, 155, 156, 159, 161, 163, 165, 179, 181, 185, 187, 189, 195, 196,198, 202, 204, 205, 207, 209, 215, 223, or a biologically activefragment or variant of any one or more thereof.
 12. An isolated and/orexogenous polynucleotide encoding at least one peptide of claim
 11. 13.A vaccine comprising the agent of claim 1, and a pharmaceuticallyacceptable carrier.
 14. (canceled)
 15. An isolated antigen presentingcell comprising the agent of claim
 1. 16. (canceled)
 17. A method ofmodulating a T cell response to a gluten peptide, inducing immunetolerance to a gluten peptide, treating celiac disease, or modifyingcytokine secretion in a subject who is sensitive to gluten, comprisingadministering to the subject an effective amount of the agent ofclaim
 1. 18-23. (canceled)
 24. A method for diagnosing celiac disease ina subject, the method comprising contacting a sample from the subjectwith the agent of claim 1, and determining in vitro whether one or moreof the peptides bind T cells in the sample, wherein the binding of oneor more of the peptides to T cells indicates that the subject has, or issusceptible to, celiac disease.
 25. (canceled)
 26. The method of claim24, wherein the method further comprises determining the efficacy ofadministering the composition to the subject.
 27. A kit comprising theagent of claim 1, and means to detect binding of one or more peptides toT cells.
 28. A method for producing an antigen presenting cell, themethod comprising i) obtaining an antigen presenting cell, and ii)contacting the cell in vitro with the agent of claim
 1. 29. (canceled)30. A method of preparing the vaccine of claim 13, the method comprisingcombining the first, second and third peptides, and optionally one ormore additional peptides comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs:47, 48, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 95,102, 103, 104, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 136, 169, 170, 171, 172, 173, 174,177, 178, 179, 180, 183, 184, 187, 188, 189, 190, 191, 192, 209, 210, ora biologically active fragment or variant of any one or more thereof,with a pharmaceutically acceptable carrier, and optionally an adjuvant.31. A method of determining whether a composition or food is capable ofcausing celiac disease, the method comprising detecting the presence ofthe agent of claim 1 in the composition or a food sample.
 32. A methodof identifying a protease that can cleave a peptide as defined in claim1, the method comprising contacting the peptide with a protease underconditions to effect specific cleavage of the peptide to produce aproteolytic product and detecting the proteolytic product produced. 33.A method for improving the half life and/or bioavailability of a peptidewhen administered to a subject, the method comprising modifying the Nterminus of the peptide to include an N terminal acetyl or pyroglutamateand modifying the C terminus of the peptide to include a C terminalamide.
 34. The method of claim 33 in which the peptide is foradministering to a subject to induce immune tolerance.
 35. A method ofmodulating a T cell response to a gluten peptide, inducing immunetolerance to a gluten peptide, treating celiac disease, or modifyingcytokine secretion in a subject who is sensitive to gluten, comprisingadministering to the subject an effective amount of the composition ofclaim 15.